Nucleic acids and proteins of a rat ganglioside GM1-specific alpha 1-2 fucosyltransferase and uses thereof

ABSTRACT

A rat ganglioside GM 1 -specific α1→2fucosyltransferase is disclosed. Nucleotide sequences of a rat ganglioside GM 1 -specific α1→2fucosyltransferase, amino acid sequences of its encoded protein (including peptide or polypeptide), and derivatives thereof are described. Also described are fragments (and derivatives and analogs thereof) which comprise a domain of rat ganglioside GM 1 -specific α1→2fucosyltransferase with catalytic activity. Methods of production of rat ganglioside GM 1 -specific α1→2fucosyltransferase and derivatives and analogs thereof (e.g. by recombinant means) are provided. Methods of inhibiting the function of rat ganglioside GM 1 -specific α1→2fucosyltransferase (e.g. by means of antisense RNA) are provided. Methods of commercial scale use of the rat ganglioside GM 1 -specific α1→2fucosyltransferase in the production of fucosyl-saccharide compositions are described. Applications of these compositions, e.g. as additives for human nutritive compositions or immunotherapeutics for cancer, are disclosed.

[0001] This invention was made with government support under ResearchGrant CA70740 from the National Cancer Institute. The government hascertain rights in the invention.

1. FIELD OF THE INVENTION

[0002] The present invention relates to a rat ganglioside GM₁-specificα1→2fucosyltransferase. The invention provides novel nucleotidesequences of a rat α1→2fucosyltransferase specific for a carbohydratemoiety found in ganglioside GM₁, more particularly, specific for aterminal galactose β1→3N-acetylgalactosamine (Galβ1→3GalNAc) saccharide,amino acid sequences of its encoded protein (including peptide orpolypeptide), and derivatives and analogs thereof. Merely for the easeof description, the enzyme is herein referred to as “GM₁-specific” or“ganglioside GM₁-specific”. The invention also relates to fragments (andderivatives and analogs thereof) which comprise a domain of ratganglioside GM₁-specific α1→2fucosyltransferase with catalytic activity.Methods of production of rat ganglioside GM₁-specificα1→2fucosyltransferase and derivatives and analogs thereof (e.g. byrecombinant means) are provided. In addition, the invention relates tomethods of inhibiting the function of rat ganglioside GM₁-specificα1→2fucosyltransferase (e.g. by means of antisense RNA). The inventionfurther relates to use of rat ganglioside GM₁-specificα1→2fucosyltransferase in the preparative production of fucosyl-GM₁.Applications of fucosyl-GM₁, for example as an immunotherapeutic forcancer, are disclosed.

2. BACKGROUND OF THE INVENTION

[0003] Citation of a reference herein shall not be construed as anadmission that such reference is prior art to the present invention.

[0004] 2.1. Fucosyltransferases

[0005] Fucosyltransferases are enzymes that catalyze the addition of afucose residue to a terminal galactose acceptor of saccharideprecursors. Fucosyltransferase activity is involved in the production ofoligosaccharides, glycolipids or glycoproteins. There are four knownclasses of fucosyltransferases, namely those that catalyze the additionof fucose in α1→2, α1→3, α1→4 and α1→6 linkages.

[0006] Fucosyltransferases are best known for their roles in thesynthesis of the oligosaccharide moieties that comprise blood groupantigenic determinants. For example, the fucosyltransferase encoded bythe H gene catalyzes the transfer of fuicose in an α1→2 linkage to theterminal galactose of Gal(β1→4)GlcNAc(β1-3)Gal-R to produce ‘Hsubstance’ on the surface of erythrocytes. Further addition ofN-acetylgalactosamine or galactose leads to the formation of the type Aor type B blood group substances respectively. An analogous enzymeencoded by the Se locus catalyzes the formation of ‘H substance’ inepithelial tissues for secretion rather than presentation at the cellsurface (Rosen et al., 1989, Dictionary of Immunology, Stockton Press,New York, pp. 1-3).

[0007] Previous experiments with H35 hepatoma cell extracts demonstratedthat transfer of fucose to neolacto-series acceptors occurred at a rateonly 2% of that found for GM₁ (Holmes, E. H., et al, 1983, J. Biol.Chem, 258:3706-3713). This substrate specificity is more restrictedcompared to other cloned α1→2fucosyltransferases but is most closelyrelated to secretor-type enzymes (Larsen, R. D., et al., 1990, Proc.Natl. Acad. Sci. USA 87:6674-6678; Kelly, R. J., et al., 1995, J. Biol.Chem. 270:46404649; Hitoshi, S., et al., 1995, J. Biol. Chem.270:8844-8850; Hitoshi, S., et al., 1996, J. Biol. Chem.271:16975-16981).

[0008] 2.2. Structure of α1→2Fucosyltransferases

[0009] To date, a number of genes encoding H-type and Se-typeα1→2fucosyltransferases have been cloned from several species oforganisms. Three human α1→2fucosyltransferases (Larsen et al., 1990,Biochemistry 87:6674-6678; Koda et al., 1997, Eur. J. Biochem.246:750-755; Kelly et al., 1995, J. Biol. Chem. 270:4640-4649), threerabbit α1→2fucosyltransferases (known as RFT-I, RFT-II and RFT-III)(Hitoshi et al., 1995, J. Biol. Chem. 270:8844-8850; Hitoshi et al.,1996, J. Biol. Chem. 271:16975-19681), and two mouseα1→2fucosyltransferases (Tsuji, 1996, GenBank accession no. Y09882; Linet al., 1998, GenBank accession no. AF064792) have been described. Piauet al. (1994, Eur. J. Biochem. 300:623-626) disclose fragments,designated FTA and FTB, of two rat α1→2fucosyltransferases isolated fromrat PROb colon adenocarcinoma cells. Piau et al. showed that antisenseexpression of the FTA or FTB nucleic acid fragments inhibited theendogenous α1→2fucosyltransferase activity of PROb cells with respect tothe synthetic fucose acceptor phenyl β-D-galactopyranoside; however theFTB fragment was not shown to be sufficient for α1→2fucosyltransferasecatalytic activity, nor was the substrate specificity of the PROb a 12fucosyltransferase activity determined.

[0010] H-type α1→2fucosyltransferases are membrane localized whereasSe-type α1→2fucosyltransferases are localized to the Golgi apparatus.Amino acid sequence alignment of membrane bound H-typeα1→2fucosyltransferases reveals that, like other glycosyltransferases,there exists a homologous domain structure comprising a shortintracellular N-terminal domain, a transmembrane domain, anextracellular stem region not required for enzymatic activity, andfinally, the catalytic domain at the C-terminus. Generally, there islittle sequence homology outside the catalytic domain.

[0011] 2.3. Ganglioside GM₁ and its Fucosylated Derivative Fucosyl-GM₁

[0012] Gangliosides are cell surface constituents comprisingglycosphingolipids (produced by the linking of ceramides tooligosaccharides) with sialic acid residues. Depending on the number ofsialic acid residues they possess, gangliosides are known as mono-, di-,tri- or polysialogangliosides. GM₁ stands for ganglioside mono(sialicacid)₁.

[0013] Fucosyl-GM₁, detected by monoclonal antibodies, is found largelyin the nervous system, and in particular on a subpopulation of neuronsin the dorsal root ganglia and dorsal horn of the spinal cord, as wellas on surrounding satellite cells surrounding the fucosyl-GM₁ positiveneurons (Kusunoki et al., 1989, Brain Res. 494:391-395; Kusonoki et al.,1992, Neurosci. Res. 15: 74-80).

[0014] Gangliosides have long been implicated in diseased states. Theyare often prominent cell surface constituents of transformed cells (seeSection 2.5, infra) and alterations in their metabolism give rise todiseases of the nervous system. For example, several fatal hereditarydiseases are caused by lysosomal storage of gangliosides wherein theabsence or deficiency of lysosomal enzymes results in the deleteriousaccumulation of gangliosides. The most well known of these diseases isthe neurodegenerative Tay-Sachs disease, which is characterized by theaccumulation of ganglioside GM₂. Accumulation of GM₁ results in GM₁Gangliosidosis.

[0015] 2.4. Regulation of Fucosyltransferase Expression

[0016] ‘H substance’, the fucosylated precursor of blood groupdeterminants, is strictly regulated temporally and spatially duringvertebrate development (Fenderson et al., 1986, Dev. Biol. 114:12-21).

[0017] Dramatic changes in the expression of cell surface glycolipidsare found with oncogenesis (Hakomori, 1989, Adv. Cancer Res. 52:257-331;Alhadeff, 1989, CRC Crit. Rev. Oncol./Hematol. 9:37-107). These changesfrequently are oncofetal in nature in that a particular carbohydratestructure may be expressed during normal fetal development, disappear inadult tissues, and reappear in association with oncogenesis giving riseto a premalignant or malignant marker. One such example is expression ofthe ganglio-B determinant (II³NeuAcIV³ αGalIV²FucGg₄) during earlystages of chemical carcinogenesis in rat liver withN-2-acetylaminofluorene (AAF) (Holmes and Hakomori, 1982, J. Biol. Chem.257:7698-7703; Scribner et al., 1983, Environ. Health Perspect.49:81-89). Expression of this determinant has been shown to be aproperty of liver parenchymal cells resulting from a carcinogenicstimulus but not hepatotoxicity (Holmes, 1990, Carcinogenesis 11:89-94).This determinant has also been shown to be developmentally regulated inrat stomach (Bonhours et al., 1987, J. Biol. Chem. 258:3706-3713).Expression of this antigen is due to the activation of anα1→2fucosyltransferase which is normally unexpressed in adult rat liverparenchymal cells. This enzyme efficiently transfers fucose onto theterminal galactose residue of a GM₁ precursor, producing fucosyl-GM₁(IV³NeuAcIV²FucGgOse₄Cer). Fucosyl-GM₁ is a substrate for aconstituitively expressed α1→3galactosyltransferase forming the bloodgroup B determinant on a ganglioside core chain (Holmes and Hakomori,1983, J. Biol. Chem. 258:3706-3713; Holmes and Hakomori, 1987, J.Biochem. 258:3706-3713). This α1→3galactosyltransferase behaves as ablood group B transferase in that it efficiently catalyzes transfer ofgalactose in α1→3-linkage to terminal galactose residues ofα1→2fucosylated neolacto- and ganglio-series acceptors (Holmes andHakomori, 1983, J. Biol. Chem. 258:3706-3713).

[0018] High α1→2fucosyltransferase expression is observed in rathepatoma H35 cells (Holmes and Hakomori, 1983, J. Biol. Chem.258:3706-3713; Holmes and Hakomori, 1987, J. Biochem. 258:3706-3713).The enzyme from H35 cells has specificity for a ganglio-series corechain. These cells accumulate large amounts of fucosyl-GM₁ (Baumann, H.,et al., 1979, Cancer Res. 39:2637-2643). Enzymological studies indicatedthis enzyme was inhibited by a wide variety of detergents, an unusualproperty for a membrane bound glycosyltransferase (Holmes, E. H., et al,1983, J. Biol. Chem, 258:3706-3713). This property may reflect a rolefor membrane phospholipids in maintaining the enzyme in an activeconformation (Holmes and Hakomori, 1987, J. Biochem. 101:1095-1105).Later studies demonstrated that active enzyme could be solubilized fromH35 cell membranes by 0.4% CHAPSO which bound to the affinity resinGDP-hexanolamine-Sepharose (Holmes, E. H., et al., 1987, J. Biochem.101:1095-1105).

[0019] Further, the observation about the production by transformedcells of high levels of fucosyl-GM₁ as a result ofα1→2fucosyltransferase activity, is not restricted to rat hepatomacells. For example, in humans, fucosyl-GM₁ is associated with small celllung carcinoma (Fredman et al., 1986, Biochim. Biophys. Acta875:316-323; Nilsson et al., 1984, Gtycoconjugate J. 1:43-49).

[0020] Generally, enzymatic oligosaccharide synthesis (includingsynthesis of glycolipids, glycoproteins, etc.) has been limited by thedifficulty of isolation and enrichment of glycosyltransferases fromnatural sources. Thus, there is a need for methods to produce easilyisolatable quantities of glycosyltranferases with high enzymaticactivity. Such glycosyltransferases, produced, e.g. in vitro, would beusefull reagents in compensating for the lack of natural resources. Inparticular, there is a need for methods to produce easily isolatableGM₁-specific α1→2fucosyltransferase. The ability to synthesizefucosyl-GM₁ in vitro is of particularly high value, as the gangliosideis important for the development of the mammalian nervous system.GM₁-specific α1→2fucosyltransferase can be used to catalyze the additionof fucose residues to terminal Galβ1→3GalNAc saccharide chains ofglycoproteins, glycolipids, glycolipoproteins and oligosaccharides,producing saccharide compositions that are useful nutritional additivesor bases therefor. Further, fucosyl-GM₁ is envisaged to be an importanttool in cancer therapy and cancer diagnostics. Until the cloning andcharacterization of the nucleic acid and amino acid sequences of thecatalytic domain and the fill length α1→2fucosyltransferase of thepresent invention, no α1→2fucosyltransferases with GM₁ specificity hadbeen identified.

3. SUMMARY OF THE INVENTION

[0021] The present invention provides a rat ganglioside GM₁-specificα1→2fucosyltransferase. As indicated above, the novel nucleic acids ofthe invention encode an α1→2fucosyltransferase enzyme specific for aterminal Galβ1→3GalNAc saccharide found naturally in ganglioside GM₁.According to the present invention, the novel nucleic acids encode anα1→2fucosyltransferase enzyme specific for the terminal Galβ1→3GalNAcmoiety which can be a part of a glycoprotein, a glycolipid, aglycolipoprotein or free oligosaccharide or polysaccharide molecule.Merely for ease of description, and not limitation, the enzyme isreferred to herein as “GM₁-specific” or “ganglioside GM₁-specific”. Moreparticularly, the invention encompasses nucleotide sequences of a ratganglioside GM₁-specific α1→2fucosyltransferase, amino acid sequences ofits encoded protein (including peptide or polypeptide), and derivativesand analogs thereof. The invention further encompasses fragments (andderivatives and thereof) which comprise a domain of rat gangliosideGM₁-specific α1→2fucosyltransferase with catalytic activity. Methods ofproduction of rat ganglioside GM₁-specific α1→2fucosyltransferase (e.g.by recombinant means), and derivatives and thereof, are provided.Methods of inhibiting the function of ganglioside GM₁-specificα1→2fucosyltransferase (e.g. by means of antisense RNA) are provided.The invention further encompasses methods for the use of rat gangliosideGM₁-specific α1→2fucosyltransferase in the production of glycoproteins,glycolipids, glycolipoproteins and free oligo- or polysaccharides.Examples of uses of these products, such as uses as nutritionaladditives, are provided. The methods are particularly useful as they canbe used in preparative biosynthesis of these saccharide-containingcompositions, and are adaptable to such synthesis in large or commercialscale production. Of particular importance is the synthesis offucosyl-GM₁, which is useful as an immunotherapeutic against cancer andneurological disease.

[0022] This invention provides an isolated or purified proteincomprising an amino acid sequence as depicted in FIG. 5 (SEQ ID NO:8).The invention further provides an isolated or purified proteincomprising amino acids 28-380 of SEQ ID NO:8 as depicted in FIG. 3A (SEQID NO:10).

[0023] This invention provides an isolated or purified proteinconsisting of an amino acid sequence as depicted in FIG. 5 (SEQ IDNO:8).

[0024] The invention further provides an isolated or purified proteinconsisting of amino acids sequence numbers 28-380 of SEQ ID NO:8 asdepicted in FIG. 3A (SEQ ID NO:10).

[0025] This invention provides an isolated or purified protein, theamino acid sequence of which consists of a catalytic domain defined byamino acid numbers 1-353 as depicted in FIG. 3A (SEQ ID NO: 10) or aminoacid numbers 28-380 as depicted in FIG. 5 (SEQ ID NO:8).

[0026] This invention provides an isolated or purified protein, theamino acid sequence of which consists of amino acid numbers 1-380 asdepicted in FIG. 5 (SEQ ID NO:8) covalently linked to at least a portionof a second protein, which second protein is not said protein defined bythe amino acid sequences as depicted in FIG. 5 (SEQ ID NO:8). In anotherembodiment, the protein is fused by a covalent bond to at least aportion of a second protein, wherein said portion is the IgG bindingdomain of protein A.

[0027] This invention provides an isolated or purified protein, theamino acid sequence of which consists of amino acids numbers 28-380 asdepicted in FIG. 5 (SEQ ID NO:8) or amino acids numbers 1-353 asdepicted in FIG. 3A (SEQ ID NO:10) covalently linked to at least aportion of a second protein, which second protein is not said proteindefined by the amino acid sequences as depicted in FIG. 5(SEQ ID NO:8).In another embodiment, the protein is fused by a covalent bond to atleast a portion of a second protein, wherein said portion is the IgGbinding domain of protein A.

[0028] This invention provides an isolated nucleic acid comprising anucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7).

[0029] This invention provides an isolated nucleic acid comprising anucleotide sequence encoding an amino acid sequence as depicted in FIG.5 (SEQ ID NO:8).

[0030] This invention provides an isolated nucleic acid comprising anucleotide sequence as depicted in FIG. 3A (SEQ ID NO:9).

[0031] This invention provides an isolated nucleic acid comprising anucleotide sequence encoding an amino acid sequence as depicted in FIG.3A (SEQ ID NO:10).

[0032] This invention provides an isolated RNA molecule comprising anucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7), wherein thebase U(uracil) is substituted for the base T (thymine) of said sequence.

[0033] This invention provides an isolated RNA molecule comprising anucleotide sequence encoding an amino acid sequence as depicted in FIG.5 (SEQ ID NO:8).

[0034] This invention provides an isolated RNA molecule comprising anucleotide sequence as depicted in FIG. 3A (SEQ ID NO:9), wherein thebase U(uracil) is substituted for the base T (thymine) of said sequence.

[0035] This invention provides an isolated RNA molecule comprising anucleotide sequence encoding an amino acid sequence as depicted in FIG.3A (SEQ ID NO:10).

[0036] This invention provides an isolated nucleic acid comprising anucleotide sequence that is the reverse complement of a nucleotidesequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ IDNO:8).

[0037] This invention provides an isolated nucleic acid comprising anucleotide sequence that is the reverse complement of a nucleotidesequence encoding an amino acid sequence as depicted in FIG. 3A (SEQ IDNO:10).

[0038] This invention provides a vector comprising (a) a nucleotidesequence as depicted in FIG. 5 (SEQ ID NO:7)and (b) an origin ofreplication. In one embodiment, the nucleotide sequence is operablylinked to a heterologous promoter.

[0039] This invention provides a vector comprising (a) a nucleotidesequence as depicted in FIG. 3A (SEQ ID NO:9)and (b) an origin ofreplication. In one embodiment, the nucleotide sequence is operablylinked to a heterologous promoter. This invention provides a vectorcomprising (a) a nucleotide sequence that is the reverse complement toall or a fragment of the nucleotide sequence as depicted in FIG. 5 (SEQID NO:7) and (b) an origin of replication. In one embodiment, thenucleotide sequence is operably linked to a heterologous promoter.

[0040] This invention provides a vector comprising (a) a nucleotidesequence that is the reverse complement to all or a fragment of thenucleotide sequence as depicted in FIG. 3A (SEQ ID NO:9) and (b) anorigin of replication. In one embodiment, the nucleotide sequence isoperably linked to a heterologous promoter.

[0041] The invention provides a vector comprising (a) a nucleotidesequence encoding an amino acid sequence as depicted in FIG. 5 (SEQ IDNO:8) and (b) an origin of replication.

[0042] The invention provides a vector comprising (a) a nucleotidesequence encoding an amino acid sequence as depicted in FIG. 3A (SEQ IDNO:10) and (b) an origin of replication.

[0043] The invention provides a recombinant cell containing arecombinant nucleic acid vector comprising a nucleotide sequence asdepicted in FIG. 5 (SEQ ID NO:7). In one embodiment, the recombinantcell is a eukaryotic cell and preferably a mammalian cell.

[0044] The invention provides a recombinant cell containing arecombinant nucleic acid vector comprising a nucleotide sequence asdepicted in FIG. 3A (SEQ ID NO:9). In one embodiment, the recombinantcell is a prokaryotic cell and preferably a bacterial cell.

[0045] This invention provides a method of producing a ratα1→2fucosyltransferase protein comprising: (a) culturing a recombinantcell containing a vector comprising a recombinant nucleotide sequence asdepicted in FIG. 5 (SEQ ID NO:7), such that the α1→2fucosyltransferaseprotein, encoded by SEQ ID NO:7, is expressed by the cell; and (b)recovering the expressed protein or a cellular fraction containing saidprotein. In one embodiment, the invention provides the purified proteinproduced by the method. In another embodiment, the invention provides acellular fraction with said protein activity.

[0046] This invention provides a method of producing a ratα1→2fucosyltransferase protein comprising: (a) culturing a recombinantcell containing a vector comprising a recombinant nucleotide sequence asdepicted in FIG. 3A (SEQ ID NO:9), such that the α1→2fucosyltransferaseprotein, encoded by SEQ ID NO:9, is expressed by the cell; and (b)recovering the expressed protein or a cellular fraction containing saidprotein. In one embodiment, the invention provides the purified proteinproduced by the method. In another embodiment, the invention provides acellular fraction with α1→2fucosyltransferase protein activity.

[0047] This invention provides a method of producing a ratα1→2fucosyltransferase protein comprising: (a) culturing a recombinantcell containing a vector comprising a recombinant nucleotide sequenceencoding a protein sequence as depicted in FIG. 5 (SEQ ID NO:8), suchthat the α1→2fucosyltransferase protein, encoded by SEQ ID NO:7, isexpressed by the cell; and (b) recovering the expressed protein or acellular fraction containing said protein. In one embodiment, theinvention provides the purified protein produced by the method. Inanother embodiment, the invention provides a cellular fraction withα1→2fucosyltransferase protein activity.

[0048] This invention provides a method of producing a ratα1→2fucosyltransferase protein comprising: (a) culturing a recombinantcell containing a vector comprising a recombinant nucleotide sequenceencoding a protein sequence as depicted in FIG. 3A (SEQ ID NO:10), suchthat the α1→2fucosyltransferase protein, encoded by SEQ ID NO:9, isexpressed by the cell; and (b) recovering the expressed protein or acellular fraction containing said protein. In one embodiment, theinvention provides the purified protein produced by the method. Inanother embodiment, the invention provides a cellular fraction withα1→2fucosyltransferase protein activity.

[0049] This invention provides a method for detecting the onset of livercancer comprising the detection of the expression of a nucleotidesequence as depicted in FIG. 5 (SEQ ID NO:9) or a fragment or complementthereof.

[0050] This invention provides a method to suppress or inhibit from acell the function of the protein of the invention, which methodcomprises contacting said cell with a nucleic acid comprising anucleotide sequence that is the reverse complement of a nucleotidesequence as depicted in FIG. 5 (SEQ ID NO:7) or a fragment thereof, oras depicted in FIG. 3A (SEQ ID NO:9) or a fragment thereof, and whereinwhen said nucleic acid is RNA, the base T (thymine) in SEQ ID NO:7 andSEQ ID NO:9 is substituted by the base U (uracil). In one embodiment,said nucleic is contained within an adenoviral or retroviral vector. Inanother embodiment, the cell is a human small cell lung carcinoma cell.

[0051] The invention provides methods for the preparative synthesis ofcompositions comprising Fucα1→2Galβ1→3GalNAc, said methods comprisingcontacting isolated or purified rat α1→2fucosyltransferase or a cellularfraction containing α1→2fucosyltransferase with GDP-fucose and amolecule having a terminal Galβ1→3GalNAc moiety. The molecule having aterminal Galβ1→3GalNAc moiety can be a glycolipid, a glycoprotein, aglycolipoprotein or a free saccharide.

[0052] Thus, the invention provides methods for the preparativesynthesis of glycolipids, glycoproteins, glycolipoproteins or freeoligosaccharides comprising Fucα1→2Galβ1→3GalNAc. In one embodiment, thefucosyl-glycolipid, -glycoprotein, -glycolipoprotein or -freeoligosaccharide or -polysaccharide produced by the method of theinvention is used as an additive to a nutritional formula.

[0053] In a particular embodiment, the invention provides a method forthe preparative synthesis of fucosyl-GM₁ comprising contacting isolatedor purified rat α1→2fucosyltransferase or a cellular fraction containingα1→2fucosyltransferase with GDP-fucose and the ganglioside GM₁ andrecovering fucosyl-GM₁.

[0054] The invention provides methods for the use of fucosyl-GM₁ inimmunotherapy for human disease comprising administering said compoundto a human patient with a disease. In one embodiment, the disease iscancer or neurological disease. In a specific preferred embodiment, saidpatient has small cell lung carcinoma.

[0055] 3.1. Abbreviations

[0056] As used herein, the following abbreviations shall have themeanings indicated.

[0057] AAF: N-2-acetylaminofluorine

[0058] α1→2FucT: α1→2fucosyltransferase

[0059] cDNA: complementary DNA

[0060] FucT, fucosyltransferase

[0061] fucosyl-GM₁:II³NeuAcIV³FucGg₄, Fucα1→2Galβ1→3

[0062] GalNAcβ1→4[NeuAcα2→3]Galβ1→4Glcβ1→1 Cer

[0063] ganglio-B: II³NeuAcIV³αGalIV²FucGg₄, Galα1→3[Fucα1→2]

[0064] Galβ1→3GalNAcβ1→4[NeuAcα2→3]Galβ1→4Glcβ1→1Cer

[0065] GM₁:II³NeuAcGg₄alβ1→3GalNAcβ1→4[NeuAcα2→3]Galβ1→4Glcβ1→1Cer

[0066] nLc₄: lactoneotetraosylceramide or

[0067] Galβ1→4GcNAcβ1→3Galβ1→4Glcβ1→1Cer

[0068] PCR: polymerase chain reaction

[0069] RT-PCR: reverse transcription—polymerase chain reaction

4. BRIEF DESCRIPTION OF THE FIGURES

[0070]FIG. 1. Portions of aligned nucleotide sequences of human (SEQ IDNO.'s:12-20) and rabbit (SEQ ID NO.'s:21-29) α1→2FucT nucleic acids. Theregions corresponding to forward and reverse primers used in the Exampledescribed infra in Section 6 are indicated except for Primer III (SEQ IDNO:3) which corresponds to the most 3′ end of the open reading frame.

[0071]FIG. 2. RT-PCR analysis of rat hepatoma H35 cell total RNA. Lane1, RT-PCR product generated using primers I (SEQ ID NO:1) and II (SEQ IDNO:2); lane 2, RT-PCR product generated using primers I (SEQ ID NO:1)and III (SEQ ID NO:3); lane 3, RT-PCR product generated using primers V(SEQ ID NO:5) and III (SEQ ID NO:3). Seven μl of each PCR mix waselectrophoresed in a 0.8% agarose gel in 1× TBE buffer. The gel wasstained with ethidium bromide. Size standards of 1.0, 0.75, and 0.5 kbare indicated.

[0072] FIGS. 3(A-B). Nucleotide (SEQ ID NO:9) and deduced amino acidsequence (SEQ ID NO:10) of the catalytic domain of rat hepatoma H35 cellα1→2FucT.

[0073]FIG. 3A. Nucleotide and deduced amino acid sequence of the 1068-bprat hepatoma H35 cell α1→2 FucT RT-PCR product generated with primers V(SEQ ID NO:5) and III (SEQ ID NO:3). The sequence extends from thesecond C residue following the EcoRI site in primer V through the end ofprimer III (SEQ ID NO:3). This nucleotide sequence has been deposited inGenBank with the Accession No. AF042743. The sequence is translated inreading frame 1. Potential N-linked glycosylation sites are shaded. Theregion which overlaps rat FTB is indicated by a solid line over thesequence. The amino acid differing between the H35 cell sequence andthat predicted by the rat FTB sequence is underlined. The stop codon isindicated in bold lettering.

[0074]FIG. 3B. Comparison of amino acid sequence homology between thecatalytic domain of rat hepatoma H35 cell α1→2FucT and human Sec2 (SEQID NO:11).

[0075]FIG. 4. TLC analysis of reaction products from transfer of[“¹⁴C]fucose to GM₁ and nLc₄ catalyzed by the pPROTA-expressed catalyticdomain of rat hepatoma H35 cell α1→2FucT. Lanes 1 and 3 show resultsfrom pPROTA expressed H35 cell α1→2FucT in the forward orientation.Lanes 2 and 4 show results from pPROTA expressed H35 cell α1→2FucT inthe reverse orientation. Lanes 1 and 2, transfer to GM₁; lanes 3 and4,transfer to nLc₄. The arrow indicates the TLC mobility of standardfucosyl-GM₁. The solvent system was composed of CHCl₃:CH₃OH:H₂O(60:40:9), containing 0.02% CaCl₂.2H₂O. See, infra, Section 6 fordetails.

[0076]FIG. 5. Nucleotide (SEQ ID NO:7) and deduced amino acid sequence(SEQ ID NO:8) of the 1140 bp rat hepatoma H35 cell α1→2FucT RT-PCRproduct generated with primers VI (SEQ ID NO:6) and III (SEQ ID NO:3).The entire coding region of 380 amino acids through the stop codon isrepresented. Potential N-linked glycosylation sites are highlighted. Theregion which was found to overlap rat FTB is indicated by a solid lineover the sequence. The amino acid differing between the H35 sequence andthat predicted by the rat FTB is underlined. Theintra-cellular/transmembrane domain comprised of 81 nucleotides (27amino acids), is shown in larger italic font.

[0077]FIG. 6. TLC analysis of reaction products from transfer of [¹⁴C]to GM₁ catalyzed by expressed recombinant full length rathepatomα1→2FucT. Lane A: transfer to GM₁ in absence of detergent orphospholipid; Lane B: transfer to GM₁ in the presence ofphosphatidylglycerol (PPG), Lane C: transfer to GM₁ in the presence ofPPG and G3634A detergent, and Lane D: transfer to GM₁ in the presence ofCHAPSO detergent. The reactions were conducted for two hours at 37° C.GM₁ standard is indicated. The solvent system was composed ofCHCl₃:CH₃OH:H₂O (60:40:9), containing 0.02% CaCl₂.2H₂O. See, infra,Section 7 for details.

[0078]FIG. 7. PCR products generated using primers I (SEQ ID NO:1) andII (SEQ ID NO:2) in RT-PCR analysis total RNA from rat hepatoma H35cells and from normal rat liver tissue. RT-PCR analysis was performed onLane 1: Total RNA from rat hepatoma H35 cells, Lane 2: Total RNA fromnormal rat liver tissue, and Lane 3: Total RNA from AAF-fed rat livertissue. The arrow on right indicates location of 0.6-kb PCR product.Size markers (in kb) are indicated on left. Five μl of each PCR mix waselectrophoresed in a 0.8% agarose gel in 1× TBE buffer. The gel wasstained with ethidium bromide. See, infra, Section 8 for details.

[0079] FIGS. 8(A-B). FIG. 8A. TLC Analysis of reaction products fromtransfer of [¹⁴C] to GM₁ catalyzed by full length expressed recombinantα1→2FucT from COS-7 cells transfected with FL-RFT-pcDNA3 in the presenceof increasing concentrations of antisense FL-RFT(−)-pcDNA3. Allreactions were carried out in the presence of CHAPSO detergent.Equimolar ratios of total DNA were maintained in each transfection byincluding varying concentrations of pcDNA3 plasmid (vector minusinsert). All lanes except Lane II were transfected with 1 μg ofFL-RFT-pcDNA3. Total FL-RFT(−)-pcDNA3 transfected was as follows: LaneI—0 μg, Lane II—1.0 μg, Lane III—1.0 μg, Lane IV—2.0 μg, Lane V—3.0 μg,and Lane VI—5.0 μg. The solvent system was composed of CHCl₃:CH₃ H:H₂O(60:40:9), containing 0.02% CaClH₂2H₂O. The GM₁ standard was visualizedby spraying in 0.5% orcinol in 2 N sulfuric acid;

[0080]FIG. 8B. Percentage reduction of initial α1→2FucT activity byincreasing doses of FL-RFT(−)-pcDNA3. The major reaction product in eachlane (indicated by arrow) (see FIG. 8A) was scraped off the plate andcounted in a scintillation counter. Cpm minus background counts of 117(Lane II) and percentage reduction of initial α1→2FucT activity byincreasing doses of FL-RFT(−)-pcDNA3 are shown. See, infra, Section 9for details.

[0081]FIG. 9. Preparative in vitro biosynthesis of fucosyl-GM₁ utilizingrecombinant rat α1→2fucosyltransferase. The results demonstrate theappearance of increasing amounts of a slower migrating bandcorresponding to fucosyl-GM₁ from transfer of fucose in the α1→2-linkageto the added GM₁ acceptor with time. The enzyme is very active, yieldingalmost complete conversion to fucosyl-GM₁ after 24 to 48 hours. See,infra, Section 10 for details.

5. DETAILED DESCRIPTION OF THE INVENTION

[0082] As described herein, the inventors have discovered andcharacterized a new ganglioside GM₁-specific α1→2fucosyltransferasegene, representing the first instance in which a nucleotide sequenceencoding a fucosyltransferase with GM₁-specificity has been identified.The novel nucleotide sequence and novel encoded protein constitute veryuseful tools for the preparative synthesis of fucosyl-containingglycolipids, glycoproteins, glycolipoproteins and oligosaccharides. In aparticular embodiment, the nucleotide sequences and encoded proteins areuseful for the preparative synthesis of fucosyl-GM₁.

[0083] The present invention thus encompasses proteins encoded by andnucleotide sequences of a rat, GM₁-specific α1→2fucosyltransferase gene.The invention further encompasses derivatives and analogs of suchα1→2fucosyltransferase protein. Nucleic acids encoding such derivativesor analogs are also within the scope of the invention. Production of theforegoing proteins, e.g., by recombinant methods, is provided.

[0084] The invention also encompasses α1→2fucosyltransferase proteinderivatives and analogs which are functionally active, i.e., which arecapable of displaying catalytic activity associated with a full-lengthGM₁-specific α1→2fucosyltransferase protein. Catalytic activity isdefined as the ability to mediate the synthesis of fucosyl-GM₁ fromstarting materials consisting of the ganglioside GM₁ and the sugarnucleotide donor GDP-fucose.

[0085] For clarity of disclosure, and not by way of limitation, thedetailed description of the invention is divided into the followingsubsections which describe or illustrate certain features, embodimentsor applications of the invention.

[0086] 5.1. Isolation of Rat α1→2Fucosyltransferase Nucleic Acids

[0087] The invention relates to the nucleotide sequences of a ratGM₁-specific α1→2fucosyltransferase (hereinafter α1→2FucT). Theinvention provides isolated or purified nucleic acids comprising anα1→2FucT encoding sequence; in another embodiment, the nucleic acidscomprise the 1069 nucleotide catalytic region of an α1→2FucT sequence.Nucleic acids can be single or double stranded. The invention alsorelates to nucleic acids hybridizable to or complementary to theforegoing sequences or their reverse complements. In specific aspects,nucleic acids are provided which comprise a sequence complementary to atleast the 1069 nucleotide catalytic of an α1→2FucT gene domain, or theentire coding region.

[0088] 5.1.1. Hybridization Conditions

[0089] In a specific embodiment, a nucleic acid which is hybridizable toan α1→2FucT nucleic acid (e.g., having a sequence as set forth in SEQ IDNO:7, or to its reverse complement, or to a nucleic acid encoding anα1→2FucT derivative or analog, or to its reverse complement), underconditions of low stringency is provided. By way of example and notlimitation, procedures using such conditions of low stringency are asfollows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci.U.S.A. 78, 6789-6792). Filters containing DNA are pretreated for 6 h at40° C. in a solution containing 35% formamide, 5× SSC, 50 mM Tris-HCl(pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/mldenatured salmon sperm DNA. Hybridizations are carried out in the samesolution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2%BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and5-20×10⁶ cpm ³²P-labeled probe is used. Filters are incubated inhybridization mixture for 18-20 h at 40° C., and then washed for 1.5 hat 55° C. in a solution containing 2× SSC, 25 mM Tris-HCl (pH 7.4), 5 mMEDTA, and 0.1% SDS. The wash solution is replaced with fresh solutionand incubated an additional 1.5 h at 60° C. Filters are blotted dry andexposed for autoradiography. If necessary, filters are washed for athird time at 65-68° C. and re-exposed to film. Other conditions of lowstringency which may be used are well known in the art (e.g., asemployed for cross-species hybridizations).

[0090] In another specific embodiment, a nucleic acid which ishybridizable to an α1→2FucT nucleic acid, or its reverse complement,under conditions of high stringency is provided. By way of example andnot limitation, procedures using such conditions of high stringency areas follows. Prehybridization of filters containing DNA is carried outfor 8 h to overnight at 65° C. in buffer composed of 6× SSC, 50 mMTris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at65° C. in prehybridization mixture containing 100 μg/ml denatured salmonsperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Washing of filters isdone at 37° C. for 1 h in a solution containing 2× SSC, 0.01% PVP, 0.01%Ficoll, and 0.01% BSA. This is followed by a wash in 0.1× SSC at 50° C.for 45 min before autoradiography. Other conditions of high stringencywhich may be used are well known in the art.

[0091] In another specific embodiment, a nucleic acid which ishybridizable to an α1→2FucT nucleic acid, or its reverse complement,under conditions of moderate stringency is provided. Selection ofappropriate conditions for such stringencies is well known in the art(see e.g., Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; see also, Ausubel et al., eds., in the Current Protocols inMolecular Biology series of laboratory technique manuals, © 1987-1997,Current Protocols, © 1994-1997 John Wiley and Sons, Inc.).

[0092] Nucleic acids encoding derivatives and analogs of α1→2FucTproteins, and α1→2FucT antisense nucleic acids are additionallyprovided. As is readily apparent, as used herein, a “nucleic acidencoding a fragment or portion of an α1→2FucT protein” shall beconstrued as referring to a nucleic acid encoding only the recitedfragment or portion of the α1→2FucT protein and not the other contiguousportions of the α1→2FucT protein as a continuous sequence.

[0093] In a preferred specific embodiment, after hybridization, washconditions are as follows. Each membrane is washed two times each for 30minutes each at 45° C. in 40 mM sodium phosphate, pH 7,2, 5% SDS, 1 mMEDTA, 0.5% bovine serum albumin, followed by four washes each for 30minutes in sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA, and subsequentlyeach membrane is treated differently as described below for low, medium,or high stringency hybridization conditions. For low stringencyhybridization, membranes are not washed further. For medium stringencyhybridization, membranes are additionally subjected to four washes eachfor 30 minutes in 40 mM sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA at55° C. For high stringency hybridization, following the r washes for lowstringency, membranes are additionally subjected to four washes each for30 minutes in 40 mM sodium phosphate, pH 7.2, 1% SDS, 1 mM EDTA at 55°C., followed by four washes each for 30 minutes in sodium phosphate, pH7.2, 1% SDS, 1 mM EDTA at 65° C.

[0094] 5.1.2. Cloning Procedures

[0095] Specific embodiments for the cloning of α1→2FucT nucleic acidsfollow. For expression cloning (a technique well known in the art), anexpression library is constructed by any method known in the art. Forexample, mRNA is isolated, cDNA is made and ligated into an expressionvector (e.g., a bacteriophage derivative) such that it is capable ofbeing expressed by the host cell into which it is then introduced.Various screening assays can then be used to select for the expressedα1→2FucT product. In one embodiment, anti-α1→2FucT antibodies can beused for selection.

[0096] In another embodiment, polymerase chain reaction (PCR) is used toamplify the desired sequence in a genomic or cDNA library, prior toselection. Oligonucleotide primers representing known α1→2FucT sequencescan be used as primers in PCR. In a preferred aspect, theoligonucleotide primers represent at least part of conserved segments ofstrong homology between α1→2FucT genes of different species. Examples ofuseful primers are provided (the α1→2FucT coding regions and complementsthereof in SEQ ID NOs:1-6). The synthetic oligonucleotides may beutilized as primers to amplify sequences from a source (RNA or DNA),preferably a cDNA library, of potential interest. PCR can be carriedout, e.g., by use of a Perkin-Elmer Cetus thermal cycler and Taqpolymerase (e.g., Gene Amp™). The nucleic acid being amplified caninclude mRNA or cDNA or genomic DNA from any species. One may synthesizedegenerate primers for amplifying homologs from other species in the PCRreactions.

[0097] It is also possible to vary the stringency of hybridizationconditions used in priming the PCR reactions, to allow for greater orlesser degrees of nucleotide sequence similarity between the knownα1→2FucT nucleotide sequences and a nucleic acid homolog (or ortholog)being isolated. For cross species hybridization, low stringencyconditions are preferred. For same species hybridization, moderatelystringent conditions are preferred. After successful amplification of asegment of an α1→2FucT homolog, that segment may be cloned and sequencedby standard techniques, and utilized as a probe to isolate a completecDNA or genomic clone. This, in turn, permits the determination of thegene's complete nucleotide sequence, the analysis of its expression, andthe production of its protein product for functional analysis, asdescribed below. In this fashion, additional nucleic acids encodingα1→2FucT proteins may be identified.

[0098] The above-described methods are not meant to limit the followinggeneral description of methods by which clones of α1→2FucT genes may beobtained.

[0099] Any eukaryotic cell potentially can serve as the nucleic acidsource for molecular cloning of α1→2FucT nucleic acids. The nucleic acidsequences encoding α1→2FucT proteins may be isolated from vertebrate,mammalian, human, porcine, bovine, feline, avian, equine, canine, aswell as additional primate sources, insects (e.g., Drosophila),invertebrates, plants, etc. The DNA may be obtained by standardprocedures known in the art from cloned DNA (e.g., a DNA “library”), bychemical synthesis, by cDNA cloning, or by the cloning of genomic DNA,or fragments thereof, purified from the desired cell (see e.g., Sambrooket al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Vol. I,II, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.;Glover, ed., 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd.,Oxford, U.K.). Clones derived from genomic DNA may contain regulatoryand intron DNA regions in addition to coding regions; clones derivedfrom cDNA will contain only exon sequences. Whatever the source, thenucleic acid should be molecularly cloned into a suitable vector forpropagation of the nucleic acid sequence.

[0100] In the molecular cloning of the gene from genomic DNA, DNAfragments are generated, some of which will encode the desired gene. TheDNA may be cleaved at specific sites using various restriction enzymes.Alternatively, one may use DNase in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, as for example,by sonication. The linear DNA fragments can then be separated accordingto size by standard techniques, including but not limited to, agaroseand polyacrylamide gel electrophoresis and column chromatography.

[0101] Once the DNA fragments are generated, identification of thespecific DNA fragment containing the desired nucleic acid may beaccomplished in a number of ways. For example, if a portion of anα1→2FucT gene or its specific RNA or a fragment thereof is available andcan be purified and labeled, the generated DNA fragments may be screenedby nucleic acid hybridization to the labeled probe (Benton and Davis,1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad.Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology tothe probe will hybridize. It is also possible to identify theappropriate fragment by restriction enzyme digestion(s) and comparisonof fragment sizes with those expected according to a known restrictionmap if such is available. Further selection can be carried out on thebasis of the properties of the gene.

[0102] Alternatively, the presence of the desired nucleic acid may bedetected by assays based on the physical, chemical, or immunologicalproperties of its expressed product. For example, cDNA clones, or DNAclones which hybrid-select the proper mRNAs, can be selected andexpressed to produce a protein that has, e.g., similar or identicalelectrophoretic migration, isoelectric focusing behavior, proteolyticdigestion maps, catalytic activity, or antigenic properties as known foran α1→2FucT protein. Using an antibody to a known α1→2FucT protein,other α1→2FucT proteins may be identified by binding of the labeledantibody to expressed putative α1→2FucT proteins, e.g., in an ELISA(enzyme-linked immunosorbent assay)-type procedure. Further, using abinding protein specific to a known α1→2FucT protein, other α1→2FucTproteins may be identified by binding to such a protein (see e.g.,Clemmons, 1993, Mol. Reprod. Dev. 35:368-374; Loddick et al., 1998,Proc. Natl. Acad. Sci. U.S.A. 95:1894-1898).

[0103] An α1→2FucT nucleic acid can also be identified by mRNA selectionusing nucleic acid hybridization followed by in vitro translation. Inthis procedure, fragments are used to isolate complementary mRNAs byhybridization. Such DNA fragments may represent available, purifiedα1→2FucT DNA of another species (e.g., mouse, human).Immunoprecipitation analysis or functional assays (e.g., catalyticactivity, etc.) of the in vitro translation products of the isolatedproducts of the isolated mRNAs identifies the mRNA and, therefore, thecomplementary DNA fragments that contain the desired sequences. Inaddition, specific mRNAs may be selected by adsorption of polysomesisolated from cells to immobilized antibodies specifically directedagainst α1→2FucT protein. A radiolabeled α1→2FucT cDNA can besynthesized using the selected mRNA (from the adsorbed polysomes) as atemplate. The radiolabeled mRNA or cDNA may then be used as a probe toidentify the α1→2FucT DNA fragments from among other genomic DNAfragments.

[0104] Alternatives to isolating the α1→2FucT genomic DNA include, butare not limited to, chemically synthesizing the nucleic acid sequenceitself from a known sequence or making cDNA to the mRNA which encodesthe α1→2FucT protein. For example, RNA for cDNA cloning of the α1→2FucTgene can be isolated from cells which express the gene.

[0105] The identified and isolated nucleic acid can then be insertedinto an appropriate cloning vector. A large number of vector-hostsystems known in the art may be used. Possible vectors include, but arenot limited to, plasmids or modified viruses, but the vector system mustbe compatible with the host cell used. Such vectors include, but are notlimited to, bacteriophages such as lambda derivatives, or plasmids suchas PBR322 or pUC plasmid derivatives or the Bluescript vector(Stratagene USA, La Jolla, Calif.). The insertion into a cloning vectorcan, for example, be accomplished by ligating the DNA fragment into acloning vector which has complementary cohesive termini. However, if thecomplementary restriction sites used to fragment the DNA are not presentin the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and an α1→2FucTnucleic acid may be modified by homopolymeric tailing. Recombinantmolecules can be introduced into host cells via transformation,transfection, infection, electroporation, etc., so that many copies ofthe nucleic acid sequence are generated.

[0106] In an alternative method, the desired nucleic acid may beidentified and isolated after insertion into a suitable cloning vectorin a “shot gun” approach. Enrichment for the desired nucleic acid, forexample, by size fractionization, can be done before insertion into thecloning vector.

[0107] In an additional embodiment, the desired nucleic acid may beidentified and isolated after insertion into a suitable cloning vectorusing a strategy that combines a “shot gun” approach with a “directedsequencing” approach. Here, for example, the entire DNA sequence of aspecific region of the genome, such as a sequence tagged site (STS), canbe obtained using clones that molecularly map in and around the regionof interest.

[0108] In specific embodiments, transformation of host cells withrecombinant DNA molecules that incorporate an isolated α1→2FucT gene,cDNA, or synthesized DNA sequence enables generation of multiple copiesof the gene. Thus, the nucleic acid may be obtained in large quantitiesby growing transformants, isolating the recombinant DNA molecules fromthe transformants and, when necessary, retrieving the inserted nucleicacid from the isolated recombinant DNA.

[0109] The α1→2FucT sequences provided by the instant invention includethose nucleotide sequences encoding substantially the same amino acidsequences as found in native α1→2FucT proteins, and those encoded aminoacid sequences with functionally equivalent amino acids, as well asthose encoding other α1→2FucT derivatives or analogs, as described inbelow for α1→2FucT derivatives and analogs.

[0110] 5.2. Expression of a Rat α1→2Fucosyltransferase Coding Sequence

[0111] The nucleotide sequence coding for an α1→2FucT protein or afunctionally active analog or other derivative thereof (see Section5.6), can be inserted into an appropriate expression vector, i.e., avector which contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequence. The necessarytranscriptional and translational signals can also be supplied by thenative α1→2FucT gene and/or its flanking regions. A variety ofhost-vector systems may be utilized to express the protein-codingsequence. These include but are not limited to mammalian cell systemsinfected with virus (e.g., vaccinia virus, adenovirus, etc.); insectcell systems infected with virus (e.g., baculovirus); microorganismssuch as yeast containing yeast vectors, or bacteria transformed withbacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elementsof vectors vary in their strengths and specificities. Depending on thehost-vector system utilized, any one of a number of suitabletranscription and translation elements may be used. In yet anotherembodiment, a fragment of an α1→2FucT protein comprising one or moredomains of the α1→2FucT protein is expressed.

[0112] Any of the methods previously described for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric nucleic acid consisting of appropriatetranscriptional/translational control signals and the protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of a nucleic acid sequence encoding an α1→2FucT protein orpeptide fragment may be regulated by a second nucleic acid sequence sothat the α1→2FucT protein or peptide is expressed in a host transformedwith the recombinant DNA molecule. For example, expression of anα1→2FucT protein may be controlled by any promoter/enhancer elementknown in the art. A promoter/enhancer may be homologous (i.e. native) orherterologous (i.e. not native). Promoters which may be used to controlthe expression of α1→2FucT coding sequences include, but are not limitedto, the SV40 early promoter region (Benoist and Chambon, 1981, Nature290:304-310), the promoter contained in the 3′ long terminal repeat ofRous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpesthymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci.U.S.A. 78:1441-1445), the regulatory sequences of the metallothioneingene (Brinster et al., 1982, Nature 296:39-42), prokaryotic expressionvectors such as the β-lactamase promoter (Villa-Kamaroff et al., 1978,Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the lac promoter (DeBoeret al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25; ScientificAmerican, 1980, 242:74-94), plant expression vectors comprising thenopaline synthetase promoter region (Herrera-Estrella et al., Nature303:209-213), the cauliflower mosaic virus 35S RNA promoter (Gardner etal., 1981, Nucl. Acids Res. 9:2871), and the promoter of thephotosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrellaet al., 1984, Nature 310:115-120), promoter elements from yeast or otherfungi such as the Gal4-responsive promoter, the ADC (alcoholdehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkalinephosphatase promoter, and the following animal transcriptional controlregions, which exhibit tissue specificity and have been utilized intransgenic animals: elastase I gene control region which is active inpancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz etal., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald,1987, Hepatology 7:425-515); a gene control region which is active inpancreatic beta cells (Hanahan, 1985, Nature 315:115-122), animmunoglobulin gene control region which is active in lymphoid cells(Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444),mouse mammary tumor virus control region which is active in testicular,breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495),albumin gene control region which is active in liver (Pinkert et al.,1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control regionwhich is active in liver (Krumlaufet al., 1985, Mol. Cell. Biol.5:1639-1648; Hammer et al., 1987, Science 235:53-58), alpha1-antitrypsin gene control region which is active in the liver (Kelseyet al., 1987, Genes and Devel. 1 :161-171), beta-globin gene controlregion which is active in myeloid cells (Mogram et al., 1985, Nature315:338-340; Kollias et al., 1986, Cell 46:89-94), myelin basic proteingene control region which is active in oligodendrocyte cells in thebrain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286), and gonadotropic releasing hormone gene controlregion which is active in the hypothalamus (Mason et al., 1986, Science234:1372-1378).

[0113] In a specific embodiment, a vector is used that comprises apromoter operably linked to an α1→2FucT nucleic acid, one or moreorigins of replication, and, optionally, one or more selectable markers(e.g., an antibiotic resistance gene).

[0114] In a specific embodiment, the promoter that is operably linked tothe rat α1→2FucT nucleic acid is not the native rat α1→2FucT genepromoter (i.e. it is a heterologous promoter).

[0115] In a specific embodiment, an expression construct is made bysubcloning an α1→2FucT coding sequence into the EcoRI restriction siteof the pPROTA mammalian cell expression vector (Henion et al., 1994,Glycobiology 4:193-202). This allows for the expression of the α1→2FucTprotein product from the subclone fused to the IgG binding domain ofprotein A.

[0116] In another specific embodiment, an expression construct is madeby subcloning an α1→2FucT coding sequence into the pcDNA3 expressionvector (Invitrogen Corp., Carlsbad, Calif.). This allows for high levelexpression of the α1→2FucT protein product from the subclone.

[0117] In another specific embodiment, an expression construct is madeby subcloning an α1→2FucT coding sequence into the pichia pPIC9expression vector (Invitrogen Corp., Carlsbad, Calif.). This allows forhigh level expression of the α1→2FucT protein product from the subclone.

[0118] Expression vectors containing α1→2FucT coding sequence insertscan be identified by four general approaches: (a) nucleic acidhybridization; (b) molecular biology, (c) expression of insertedsequences; and (d) presence or absence of “marker” gene functions . Inthe first approach, the presence of an α1→2FucT nucleic acid inserted inan expression vector can be detected by nucleic acid hybridization usingprobes comprising sequences that are homologous to an inserted α1→2FucTnucleic acid. In the second approach, a combination of molecular biologyand “marker” gene function are used to identify recombinant expressionvectors containing the α1→2FucT insert. For example, if the α1→2FucTnucleic acid is inserted in the EcoRI site of the pcDNA3 vector, whichcodes for both Ampicillin and Neomycin resistance, bacterial cells thattake up the vector are identified by their resistance to Ampicillinand/or Neomycin, and those vectors containing the α1→2FucT insert can beidentified by restriction digestion of the amplified vector DNA withEcoRI. In the third approach, recombinant expression vectors can beidentified by assaying the α1→2FucT product expressed by therecombinant. Such assays can be based, for example, on the physical orfunctional properties of the α1→2FucT protein in in vitro assay systems,e.g., the catalysis of fucosyl-GM₁ synthesis. In the fourth approach,the vector/host system can be identified based upon the presence orabsence of certain “marker” gene functions (e.g., thymidine kinaseactivity, β-galactosidase, resistance to antibiotics, transformationphenotype, occlusion body formation in baculovirus, etc.) caused by theinsertion of an α1→2FucT nucleic acid in the vector. For example, if theα1→2FucT nucleic acid is inserted within the marker gene sequence of thevector, recombinants containing the α1→2FucT insert can be identified bythe absence of the marker gene function.

[0119] Once a particular recombinant DNA molecule is identified andisolated, several methods known in the art may be used to propagate it.Once a suitable host system and growth conditions are established,recombinant expression vectors can be propagated and prepared inquantity. As previously explained, the expression vectors which can beused include, but are not limited to, the following vectors or theirderivatives: human or animal viruses such as vaccinia virus oradenovirus; insect viruses such as baculovirus; yeast vectors;bacteriophage vectors (e.g., lambda phage), and plasmid and cosmid DNAvectors, to name but a few.

[0120] In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes thenucleic acid product in the specific fashion desired. Expression fromcertain promoters can be elevated in the presence of certain inducers;thus, expression of the genetically engineered α1→2FucT protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation) of proteins.Appropriate cell lines or host systems can be chosen to ensure thedesired modification and processing of the foreign protein expressed.For example, expression in a bacterial system can be used to produce asoluble α1→2FucT catalytic domain. Expression in animal cells can beused to ensure folding, proper membrane insertion and glycosylation ofα1→2FucT.

[0121] In other specific embodiments, the α1→2FucT protein, derivativeor analog may be expressed as a fusion, or chimeric protein product(comprising the protein, derivative or analog joined via a covalent bondsuch a peptide bond to a heterologous protein sequence (of a differentprotein)). A chimeric protein may include fusion of the α1→2FucTprotein, derivative or analog to a second protein or at least a portionthereof, wherein a portion is one (preferably 10, 15, or 20) or moreamino acids of said second protein. The second protein, or one or moreamino acid portion thereof, may be from a different rat α1→2FucT proteinor may be from a protein that is not a rat α1→2FucT protein. Such achimeric product can be made by ligating the appropriate nucleic acidsequences encoding the desired amino acid sequences to each other bymethods known in the art, in the proper coding frame, and expressing thechimeric product by methods commonly known in the art. Alternatively,such a chimeric product may be made by protein synthetic techniques,e.g., by use of a peptide synthesizer. In a specific embodiment, theamino acid portion of the second protein is one that allows for theextracellular secretion of the α1→2FucT catalytic domain, e.g. the Igbinding domain of protein A (Henion et al., 1994, Glycobiology4:193-202). In a specific embodiment, the amino acid portion of thesecond protein is one that allows for the membrane localization of theα1→2FucT catalytic domain, e.g. the type I transmembrane domain ofsevenless (Basler et al., 1991) or Notch (reviewed by Weinmaster, 1997,Mol. Cell. Neurosci. 9:91-102), the type II transmembrane domain ofhuman H-type α1→2fucosyltransferase (Koda et al., 1997, Eur. J. Biochem.300:623-626), or the myristylation signal of src proteins (Cross et al.,1984, Mol. Cell Biol. 4:1834-1842; Simon et al., 1985, Cell 42:831-840).

[0122] 5.3. Identification and Purification of Rat α1→2FucT Products

[0123] In particular aspects, the invention provides amino acidsequences of α1→2FucT proteins and derivatives or analogs thereof whichcomprise an antigenic determinant (i.e., can be recognized by anantibody) or which are otherwise functionally active, as well as nucleicacid sequences encoding the foregoing. “Functionally active” α1→2FucTmaterial as used herein refers to that material displaying one or morefunctional activities associated with a full-length (wild-type) α1→2FucTprotein, e.g., enzymatic ability to transfer fucose or a fucosyl moietyin an α1→2 linkage to a terminal galactose of a Galβ1→3GalNAc moiety,e.g. GM₁, with specificity, etc.

[0124] Once a recombinant nucleic acid which expresses the α1→2FucTcoding sequence is identified, the product can be analyzed. This isachieved by assays based on the physical or functional properties of theproduct, including radioactive labeling of the product followed byanalysis by gel electrophoresis, immunoassay, etc.

[0125] Once the α1→2FucT protein is identified, it may be isolated andpurified by standard methods including chromatography (e.g., ionexchange, affinity, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. The functional properties may be evaluatedusing any suitable assay (see, e.g., Section 5.7).

[0126] Alternatively, once an α1→2FucT protein produced by a recombinantis identified, the amino acid sequence of the protein can be deducedfrom the nucleotide sequence of the chimeric nucleic acid. As a result,the protein can be synthesized by standard chemical methods known in theart (e.g., see Hunkapiller et al., 1984, Nature 310:105-111).

[0127] In another alternate embodiment, native α1→2FucT proteins can bepurified from natural sources, by standard methods such as thosedescribed above (e.g., immunoaffinity purification).

[0128] In a specific embodiment of the present invention, such α1→2FucTproteins, whether produced by recombinant DNA techniques or by chemicalsynthetic methods or by purification of native proteins, include but arenot limited to those containing, as a primary amino acid sequence, allor part of the amino acid sequence substantially as depicted in FIG. 5(SEQ ID NO:8), as well as derivatives and analogs thereof, includingproteins homologous thereto.

[0129] 5.4. Structure of α1→2FucT Nucleic Acids and Proteins

[0130] The structure of α1→2FucT nucleic acids and proteins of theinvention can be analyzed by various methods known in the art. Someexamples of such methods are described below.

[0131] 5.4.1. Genetic Analysis

[0132] The cloned DNA or cDNA corresponding to an α1→2FucT nucleic acidcan be analyzed by methods including but not limited to Southernhybridization (Southern, 1975, J. Mol. Biol. 98:503-517), Northernhybridization (see e.g., Freeman et al., 1983, Proc. Natl. Acad. Sci.U.S.A. 80:4094-4098), restriction endonuclease mapping (Maniatis, 1982,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.), and DNA sequence analysis.Accordingly, this invention provides nucleic acid probes recognizing anα1→2FucT nucleic acid. For example, polymerase chain reaction (PCR; U.S.Pat. Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al., 1988,Proc. Natl. Acad. Sci. U.S.A. 85:7652-7656; Ochman et al., 1988,Genetics 120:621-623; Loh et al., 1989, Science 243:217-220) followed bySouthern hybridization with an α1→2FucT-specific probe can allow thedetection of an α1→2FucT gene in DNA from various cell types. Methods ofamplification other than PCR are commonly known and can also beemployed. In one embodiment, Southern hybridization can be used todetermine the genetic linkage of an α1→2FucT gene. Northernhybridization analysis can be used to determine the expression of anα1→2FucT gene. Various cell types, at various states of development oractivity can be tested for α1→2FucT gene expression. The stringency ofthe hybridization conditions for both Southern and Northernhybridization can be manipulated to ensure detection of nucleic acidswith the desired degree of relatedness to the specific α1→2FucT-probeused. Modifications of these methods and other methods commonly known inthe art can be used.

[0133] Restriction endonuclease mapping can be used to roughly determinethe genetic structure of an α1→2FucT nucleic acid. Restriction mapsderived by restriction endonuclease cleavage can be confirmed by DNAsequence analysis.

[0134] DNA sequence analysis can be performed by any techniques known inthe art, including but not limited to the method of Maxam and Gilbert(1980, Meth. Erzymol. 65:499-560), the Sanger dideoxy method (Sanger etal., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNApolymerase (Tabor and Richardson, U.S. Pat. No. 4,795,699), or use of anautomated DNA sequenator (e.g., Applied Biosystems, Foster City,Calif.).

[0135] 5.4.2. Protein Analysis

[0136] The amino acid sequence of an α1→2FucT protein can be derived bydeduction from the DNA sequence, or alternatively, by direct sequencingof the protein, e.g., with an automated amino acid sequencer.

[0137] An α1→2FucT protein sequence can be further characterized by ahydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci.U.S.A. 78:3824). A hydrophilicity profile can be used to identify thehydrophobic and hydrophilic regions of the α1→2FucT protein and thecorresponding regions of the gene sequence which encode such regions.

[0138] Structural prediction analysis (Chou and Fasman, 1974,Biochemistry 13:222) can also be done, to identify regions of anα1→2FucT protein that assume specific secondary structures.

[0139] Manipulation, translation, and secondary structure prediction,open reading frame prediction and plotting, as well as determination ofsequence homologies, can also be accomplished using computer softwareprograms available in the art.

[0140] Other methods of structural analysis can also be employed. Theseinclude but are not limited to X-ray crystallography (Engstom, 1974,Biochem. Exp. Biol. 11:7-13l), nuclear magnetic resonance spectroscopy(Clore and Gonenborn, 1989, CRC Crit. Rev. Biochem. 24:479-564) andcomputer modeling (Fletterick and Zoller, 1986, Computer Graphics andMolecular Modeling, in Current Communications in Molecular Biology, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

[0141] 5.5. Antibodies

[0142] According to the invention, α1→2FucT protein, its derivatives, oranalogs thereof, may be used as an immunogen to generate antibodieswhich immunospecifically bind such an immunogen. Such antibodies includebut are not limited to polyclonal, monoclonal, chimeric, single chain,Fab fragments, and an Fab expression library. In another embodiment,antibodies to a domain (e.g., an α1→2FucT receptor binding domain) of anα1→2FucT protein are produced. In aspecific embodiment, fragments of anα1→2FucT protein identified as hydrophilic are used as immunogens forantibody production.

[0143] Various procedures known in the art may be used for theproduction of polyclonal antibodies to an α1→2FucT protein or derivativeor analog. In a particular embodiment, rabbit polyclonal antibodies toan epitope of an α1→2FucT protein consisting of the sequence of SEQ IDNO:2, or a subsequence thereof, can be obtained. For the production ofantibody, various host animals can be immunized by injection with thenative α1→2FucT protein, or a synthetic version, or derivative thereof,including but not limited to rabbits, mice, rats, etc. Various adjuvantsmay be used to increase the immunological response, depending on thehost species, and including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and corynebacterium parvum.

[0144] For preparation of monoclonal antibodies directed to an α1→2FucTprotein sequence or analog thereof, any technique which provides for theproduction of antibody molecules by continuous cell lines in culture maybe used. For example, the hybridoma technique originally developed byKohler and Milstein, (Kohler and Milstein 1975, Nature 256:495-497), aswell as the trioma technique, the human B-cell hybridoma technique(Kozbor et at., 1983, Immunology Today 4:72), and the BBV-hybridomatechnique to produce human monoclonal antibodies (Cole et al., 1985, inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp.77-96). In an additional embodiment of the invention, monoclonalantibodies can be produced in germ-free animals utilizing recenttechnology (see e.g., PCT/US90/02545). According to the invention, humanantibodies may be used and can be obtained by using human hybridomas(Cole et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or bytransforming human B cells with EBV virus in vitro (Cole et al., 1985,in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96).In fact, according to the invention, techniques developed for theproduction of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl.Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing thegenes from a mouse antibody molecule specific for an α1→2FucT proteintogether with genes from a human antibody molecule of appropriatebiological activity can be used; such antibodies are within the scope ofthis invention.

[0145] According to the invention, techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778) can beadapted to produce α1→2FucT-specific single chain antibodies. Anadditional embodiment of the invention utilizes the techniques describedfor the construction of Fab′ expression libraries (Huse et al., 1989,Science 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for α1→2FucTproteins, derivatives, or analogs.

[0146] Antibody fragments which contain the idiotype of the molecule canbe generated by known techniques. For example, such fragments includebut are not limited to, the F(ab′)₂ fragment which can be produced bypepsin digestion of the antibody molecule, the Fab′ fragments which canbe generated by reducing the disulfide bridges of the F(ab′)₂ fragment,the Fab fragments which can be generated by treating the antibodymolecule with papain and a reducing agent, and Fv fragments.

[0147] In the production of antibodies, screening for the desiredantibody can be accomplished by techniques known in the art (e.g.,enzyme-linked immunosorbent assay or ELISA). For example, to selectantibodies which recognize a specific domain of a α1→2FucT protein, onemay assay generated hybridomas for a product which binds to a α1→2FucTfragment containing such domain. For selection of an antibody thatspecifically binds a first α1→2FucT homolog but which does notspecifically bind a different α1→2FucT homolog, one can select on thebasis of positive binding to the first α1→2FucT homolog and a lack ofbinding to the second α1→2FucT homolog.

[0148] Antibodies specific to a domain of an α1→2FucT protein are alsoprovided. Antibodies specific to an epitope of an α1→2FucT protein arealso provided.

[0149] The foregoing antibodies can be used in methods known in the artrelating to the localization and activity of the α1→2FucT proteinsequences of the invention, erg., for imaging these proteins, measuringlevels thereof in appropriate physiological samples, in diagnosticmethods, etc.

[0150] 5.6. α1→2FucT Proteins and Derivatives

[0151] The invention further encompasses α1→2FucT proteins, derivatives,analogs, and molecules of α1→2FucT proteins. As used herein, a moleculedefined by a particular SEQ ID NO, shall be construed to mean that thesequence of that molecule comprises that SEQ ID NO, unless explicitlyindicated otherwise to mean that the sequence of the molecule consistsof that SEQ ID NO. Nucleic acids encoding α1→2FucT protein derivativesand protein analogs are also provided. In one embodiment, the α1→2FucTproteins are encoded by the α1→2FucT nucleic acids described in Section5.1 above. In particular aspects, the proteins, derivatives, or analogsare of α1→2FucT proteins encoded by the amino acid sequence of (SEQ IDNO:8).

[0152] The production and use of derivatives and analogs related to anα1→2FucT protein are within the scope of the present invention. In aspecific embodiment, the derivative or analog is functionally active,i.e., capable of exhibiting one or more functional activities associatedwith a full-length, wild-type α1→2FucT protein. As one example, suchderivatives or analogs which have the desired immunogenicity orantigenicity can be used in immunoassays, for immunization, forinhibition of α1→2FucT activity, etc. As another example, suchderivatives or analogs which have the desired binding activity can beused for binding to the InR gene product. As yet another example, suchderivatives or analogs which have the desired binding activity can beused for binding to a binding protein specific for a known α1→2FucTprotein (see e.g., Clemmons, 1993, Mol. Reprod. Dev. 35:368-374; Loddicket al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:1894-1898). Derivativesor analogs that retain, or alternatively lack or inhibit, a desiredα1→2FucT protein property-of-interest (e.g., binding to an α1→2FucTprotein binding partner), can be used as inducers, or inhibitors,respectively, of such property and its physiological correlates. Aspecific embodiment relates to an α1→2FucT protein fragment that can bebound by an anti-α1→2FucT protein antibody. Derivatives or analogs of anα1→2FucT protein can be tested for the desired activity by proceduresknown in the art, including but not limited to the assays described inSection (5.10 and 5.11 below).

[0153] In particular, α1→2FucT derivatives can be made by alteringα1→2FucT sequences by substitutions, additions (e.g., insertions) ordeletions that provide for functionally equivalent molecules. Due to thedegeneracy of nucleotide coding sequences, other DNA sequences whichencode substantially the same amino acid sequence as an α1→2FucT nucleicacid may be used in the practice of the present invention. These includebut are not limited to nucleotide sequences comprising all or portionsof an α1→2FucT nucleic acid which is altered by the substitution ofdifferent codons that encode a functionally equivalent amino acidresidue within the sequence, thus producing a silent change. Likewise,the α1→2FucT derivatives of the invention include, but are not limitedto, those containing, as a primary amino acid sequence, all or part ofthe amino acid sequence of an α1→2FucT protein including alteredsequences in which functionally equivalent amino acid residues aresubstituted for residues within the sequence resulting in a silentchange. For example, one or more amino acid residues within the sequencecan be substituted by another amino acid of a similar polarity whichacts as a functional equivalent, resulting in a silent alteration.Substitutions for an amino acid within the sequence may be selected fromother members of the class to which the amino acid belongs. For example,the nonpolar (hydrophobic) amino acids include alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan and methionine.The polar neutral amino acids include glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine. The positively charged(basic) amino acids include arginine, lysine and histidine. Thenegatively charged (acidic) amino acids include aspartic acid andglutamic acid. Such substitutions are generally understood to beconservative substitutions.

[0154] In a specific embodiment of the invention, proteins consisting ofor comprising a fragment of an α1→2FucT protein consisting of at least10 (continuous) amino acids of the α1→2FucT protein are provided. Inother embodiments, the fragment consists of at least 20 or at least 50amino acids of the α1→2FucT protein. In specific embodiments, suchfragments are not larger than 35, 100 or 200 amino acids. Derivatives oranalogs of α1→2FucT proteins include but are not limited to thosemolecules comprising regions that are substantially homologous to anα1→2FucT protein or fragment thereof (e.g., in various embodiments, atleast 60% or 70% or 80% or 90% or 95% identity over an amino acidsequence of identical size or when compared to an aligned sequence inwhich the alignment is done by a computer homology program known in theart) or whose encoding nucleic acid is capable of hybridizing to acoding α1→2FucT gene sequence, under high stringency, moderatestringency, or low stringency conditions.

[0155] Specifically, by way of example computer programs for determininghomology may include but are not limited to TBLASTN, BLASTP, FASTA,TFASTA, and CLUSTALW (Altschul et al., 1990, J. Mol. Biol.215(3):403-10; see, Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA85(8):2444-8; Thompson, et al., 1994, Nucleic Acids Res. 22(22):4673-80;Higgins, et al., 1996, Methods Enzymol 266:383-402).

[0156] Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov)(Altschul et al., 1990, J. of Molec. Biol., 215:403-410. “The BLASTAlgorithm”; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402) is aheuristic search algorithm tailored to searching for sequence similaritywhich ascribes significance using the statistical methods of Karlin andAltschul 1990, Proc. Nat'l Acad. Sci. USA, 87:2264-68; 1993, Proc. Nat'lAcad. Sci. USA 90:5873-77. Five specific BLAST programs perform thefollowing tasks: 1) The BLASTP program compares an amino acid querysequence against a protein sequence database; 2) The BLASTN programcompares a nucleotide query sequence against a nucleotide sequencedatabase; 3) The BLASTX program compares the six-frame conceptualtranslation products of a nucleotide query sequence (both strands)against a protein sequence database; 4) The TBLASTN program compares aprotein query sequence against a nucleotide sequence database translatedin all six reading frames (both strands); 5) The TBLASTX programcompares the six-frame translations of a nucleotide query sequenceagainst the six-frame translations of a nucleotide sequence database.

[0157] Smith-Waterman (database: European Bioinformatics Institute ”wwwz.ebi.ac.uk/bic_sw/) (Smith-Waterman, 1981, J. of Molec. Biol.,147:195-197) is a mathematically rigorous algorithm for sequencealignments.

[0158] FASTA (see Pearson et al., 1988, Proc. Nat'l Acad. Sci. USA,85:2444-2448) is a heuristic approximation to the Smith-Watermanalgorithm.

[0159] For a general discussion of the procedure and benefits of theBLAST, Smith-Waterman and FASTA algorithms see Nicholas et al., 1998, “ATutorial on Searching Sequence Databases and Sequence Scoring Methods”(www.psc.edu) and references cited therein.

[0160] The α1→2FucT derivatives and analogs of the invention can beproduced by various methods known in the art. The manipulations whichresult in their production can occur at the gene or protein level. Forexample, a cloned α1→2FucT nucleic acid sequence can be modified by anyof numerous strategies known in the art (Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.). The sequence can be cleavedat appropriate sites with restriction endonuclease(s), followed byfurther enzymatic modification if desired, isolated, and ligated invitro. In the production of a modified nucleic acid encoding aderivative or analog of an α1→2FucT protein, care should be taken toensure that the modified nucleic acid remains within the sametranslational reading frame as the native protein, uninterrupted bytranslational stop signals, in the gene region where the desiredα1→2FucT protein activity is encoded.

[0161] Additionally, an α1→2FucT nucleic acid sequence can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or to form new restriction endonuclease sites or destroy preexistingones, to facilitate further in vitro modification. Any technique formutagenesis known in the art can be used, including but not limited to,chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson etal., 1978, J. Biol. Chem. 253:6551), use of TABSG linkers (Pharmacia),PCR with primers containing a mutation, etc.

[0162] Manipulations of an α1→2FucT protein sequence may also be made atthe protein level. Included within the scope of the invention areα1→2FucT protein fragments or other derivatives or analogs which aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin, etc.

[0163] In addition, analogs and derivatives of an aα1→2FucT protein canbe chemically synthesized. For example, a peptide corresponding to aportion of an α1→2FucT protein which comprises the desired domain, orwhich mediates the desired activity in vitro, can be synthesized by useof a peptide synthesizer. Furthermore, if desired, nonclassical aminoacids or chemical amino acid analogs can be introduced as a substitutionor addition into the α1→2FucT sequence. Non-classical amino acidsinclude but are not limited to the D-isomers of the common amino acids,α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid,γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β-methyl amino acids,Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

[0164] In a specific embodiment, an α1→2FucT protein derivative is achimeric or fusion protein comprising an α1→2FucT protein or fragmentthereof (preferably consisting of at least a domain or motif of theα1→2FucT protein, or at least 10 amino acids of the α1→2FucT protein)joined at its amino- or carboxy-terminus via a peptide bond to an aminoacid sequence of a different protein. In specific embodiments, the aminoacid sequence of the different protein is at least 6, 10, 20 or 30continuous amino acids of the different proteins or a portion of thedifferent protein that is functionally active. In one embodiment, such achimeric protein is produced by recombinant expression of a nucleic acidencoding the protein (comprising an α1→2FucT-coding sequence joinedin-frame to a coding sequence for a different protein). Such a chimericproduct can be made by ligating the appropriate nucleic acid sequencesencoding the desired amino acid sequences to each other by methods knownin the art, in the proper coding frame, and expressing the chimericproduct by methods commonly known in the art. Alternatively, such achimeric product may be made by protein synthetic techniques, e.g., byuse of a peptide synthesizer. Chimeric genes comprising the wholeα1→2FucT open reading frame or the nucleotides encoding the catalyticdomain fused to any heterologous protein-encoding sequences may beconstructed.

[0165] In another specific embodiment, the α1→2FucT derivative is amolecule comprising a region of homology with the full length orcatalytic domain of α1→2FucT protein. By way of example, in variousembodiments, a first protein region can be considered “homologous” to asecond protein region when the amino acid sequence of the first regionis at least 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, or 95% identical,when compared to any sequence in the second region of an equal number ofamino acids as the number contained in the first region or when comparedto an aligned sequence of the second region that has been aligned by acomputer homology program known in the art. For example, a molecule cancomprise one or more regions homologous to an α1→2FucT catalytic domain(see Section 5.6.1) or a portion thereof.

[0166] 5.7. Elimination of α1→2FucT Activity

[0167] The present invention provides for methods of creating cellslacking α1→2fucosyltransferase activity.

[0168] In one embodiment, loss-of-function phenotypes are generated byantisense RNA methods (Schubiger and Edgar, 1994, Methods in CellBiology 44:697-713). One form of the antisense RNA method involves theinjection of cells with an antisense RNA that is partially homologous tothe gene-of-interest (in this case an α1→2FucT nucleic acid). Anotherform of the antisense RNA method involves expression of an antisense RNApartially homologous to the gene-of-interest by operably joining aportion of the gene-of-interest in the antisense orientation to apowerful promoter that can drive the expression of large quantities ofantisense RNA, either generally throughout the animal or in specifictissues.

[0169] In a second embodiment, loss-of-function phenotypes are generatedby cosuppression methods (Bingham, 1997, Cell 90(3):385-7; Smyth, 1997,Curr. Biol. 7(12):793-5; Que and Jorgensen, 1998, Dev. Genet.22(1):100-9). Cosuppression is a phenomenon of reduced gene expressionproduced by expression or injection of a sense strand RNA correspondingto a partial segment of the gene-of-interest. Cosuppression effects havebeen employed extensively in plants to generate loss-of-functionphenotypes.

[0170] 5.7.1. Antisense Regulation of Gene Expression

[0171] The invention provides for antisense uses of rat α1→2FucT nucleicacids. In a specific embodiment, an α1→2FucT protein function isinhibited by use of α1→2FucT antisense nucleic acids. The presentinvention provides for use of nucleic acids of at least six nucleotidesthat are antisense to a gene or cDNA encoding an α1→2FucT protein or aportion thereof. An α1→2FucT “antisense” nucleic acid as used hereinrefers to a nucleic acid capable of hybridizing to a sequence-specific(i.e. non-poly A) portion of an α1→2FucT RNA (preferably mRNA) by virtueof some sequence complementarity. Antisense nucleic acids may also bereferred to as inverse complement nucleic acids. The antisense nucleicacid may be complementary to a coding and/or noncoding region of anα1→2FucT mRNA. Such antisense nucleic acids have utility in inhibitingan α1→2FucT protein function.

[0172] The antisense nucleic acids of the invention can beoligonucleotides that are double-stranded or single-stranded, RNA or DNAor a modification or derivative thereof, which can be directlyadministered to a cell. The α1→2FucT antisense nucleic acids of theinvention are preferably oligonucleotides (ranging from 6 to about 50oligonucleotides). In specific aspects, an oligonucleotide is at least10 nucleotides, at least 15 nucleotides, at least 100 nucleotides, or atleast 200 nucleotides in length. The oligonucleotide can be DNA or RNAor chimeric mixtures or derivatives or modified versions thereof, orsingle-stranded or double-stranded. The oligonucleotide can be modifiedat the base moiety, sugar moiety, or phosphate backbone. Theoligonucleotide may include other appending groups such as peptides, oragents facilitating transport across the cell membrane (see e.g.,Letsinger et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556;Lemaitre et al., 1987, Proc. Natl. Acad. Sci. U.S.A. 84:648-652; PCTPublication No. WO 88/09810, published Dec. 15, 1988) or the blood-brainbarrier (see e.g., PCT Publication No. WO 89/10134, published Apr. 25,1988), hybridization-triggered cleavage agents (see e.g., Krol et al.,1988, BioTechniques 6:958-976) or intercalating agents (see e.g., Zon,1988, Pharm. Res. 5:539-549).

[0173] In a preferred aspect of the invention, an α1→2FucT antisenseoligonucleotide is provided as single-stranded DNA. In another preferredaspect, such an oligonucleotide comprises a sequence antisense to thesequence encoding a B peptide domain or an A peptide domain of anα1→2FucT protein. The oligonucleotide may be modified at any position onits structure with substituents generally known in the art.

[0174] The α1→2FucT antisense oligonucleotide may comprise at least onemodified base moiety which is selected from the group including but notlimited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. In another embodiment, the oligonucleotidecomprises at least one modified sugar moiety selected from the groupincluding but not limited to arabinose, 2-fluoroarabinose, xylulose, andhexose.

[0175] In yet another embodiment, the oligonucleotide comprises at leastone modified phosphate backbone selected from the group consisting of aphosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

[0176] In yet another embodiment, the oligonucleotide is an α-anomericoligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide may beconjugated to another molecule, e.g., a peptide, ahybridization-triggered cross-linking agent, a transport agent, ahybridization-triggered cleavage agent, etc.

[0177] Oligonucleotides of the invention may be synthesized by standardmethods known in the art, e.g., by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides may be synthesizedby the method of Stein et al. (Stein et al., 1988, Nucl. Acids Res.16:3209), methylphosphonate oligonucleotides can be prepared by use ofcontrolled pore glass polymer supports (Sarin et al., 1988, Proc. Natl.Acad. Sci. U.S.A. 85:7448-7451), etc.

[0178] In a specific embodiment, an α1→2FucT antisense oligonucleotidecomprises catalytic RNA, or a ribozyme (see e.g, PCT Publication WO90/11364, published Oct. 4, 1990; Sarver et al., 1990, Science247:1222-1225). In another embodiment, the oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

[0179] In a preferred embodiment, the antisense nucleic acids of theinvention are expressed intracellularly by transcription from anexogenous sequence. For example, a vector can be introduced in vivo suchthat it is taken up by a cell, within which cell the vector or a portionthereof is transcribed, producing an antisense nucleic acid (RNA) of theinvention. Such a vector would contain a sequence encoding the α1→2FucTantisehse nucleic acid. Such a vector can remain episomal or becomechromosomally integrated, as long as it can be transcribed to producethe desired antisense RNA. The antisense nucleic acid can beadministered by use of an adenoviral or retroviral vector (see US4,980,286), by direct injection, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), by coating with lipids orcell-surface receptors or transfecting agents, or by administering it inlinkage to a homeobox-like peptide which is known to enter the nucleus(see e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA88:1864-1868), etc. Vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others known in the art, used for replication and expression inmammalian cells. Expression of the sequence encoding the α1→2FucTantisense RNA can be by any promoter known in the art. Such promoterscan be inducible or constitutive. Such promoters include but are notlimited to: the SV40 early promoter region (Benoist and Chambon, 1981,Nature 290:304-310), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797),the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of themetallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc.

[0180] The antisense nucleic acids of the invention comprise a sequencecomplementary to at least a sequence-specific portion of an RNAtranscript of an α1→2FucT gene. However, absolute complementarity,although preferred, is not required. A sequence “complementary to atleast a portion of an RNA,” as referred to herein, means a sequencehaving sufficient complementarity to be able to hybridize with the RNA,forming a stable duplex; in the case of double-stranded α1→2FucTantisense nucleic acids, a single strand of the duplex DNA may thus betested, or triplex formation may be assayed. The ability to hybridizewill depend on both the degree of complementarity and the length of theantisense nucleic acid. Generally, the longer the hybridizing nucleicacid, the more base mismatches with an α1→2FucT RNA it may contain andstill form a stable duplex (or triplex, as the case may be). One skilledin the art can ascertain a tolerable degree of mismatch by use ofstandard procedures to determine, e.g., the melting point of thehybridized complex.

[0181] 5.8. Biochemical Assays Using α1→2FucT Proteins

[0182] The functional activity of α1→2FucT proteins or derivatives canbe assayed by various methods known to one skilled in the art.

[0183] For example, as illustrated in Section 6.3.2, infra, the activityof α1→2fucosyltransferase coupled to the IgG-binding domain of Protein Acan be determined in reaction mixtures containing 2.5 μmol of HEPESbuffer, pH7.2, 30 μg of GM₁. ganglioside or nLc₄, 250 μgphosphatidylglycerol, 1 μmol of MnCl₂, 0.5 μmol of CDP-choline, 15 nmolof GDP-[¹⁴C]fucose (15,000 cpm/nmol), and bound to IgG-agarose beads ina total volume of 0.1 ml. The reaction mixtures are incubated for 2 h at37° C., terminated by the addition of 0.1 ml of CHCl₃:CH₃OH (2:1),streaked onto 4-cm-wide strips of Whatman 3 paper and developed withwater overnight. The papers are dried and the labeled product extractedfrom the origins with 2- to 5-ml washes of CHCl₃:CH₃OH:H₂O (10:5:1). Thecombined eluates are concentrated to dryness by an N₂ stream anddissolved in 20 μl of CHCl₃:CH₃OH (2:1). A 10-μl aliquot of each isspotted onto a HP-TLC plate (Merck)and developed in a solvent systemcomposed of CHCl₃:CH₃OH:H₂O (60:40:9), 0.02% CaCl₂.2H₂O. The radioactiveproducts were located by autoradiography.

[0184] 5.9. Additional Applications and Uses of α1→2FucT Nucleic Acidsand Proteins

[0185] Provided below are additional non-limiting methods of using theα1→2FucT nucleic acids and proteins of the invention.

[0186] 5.9.1. Detection of Oncogenesis

[0187] As α1→2FucT expression is often activated during oncogenictransformation (see Section 2.5 supra), oncogenic transformation of testtissues can be detected by assaying for changes in the expression ofα1→2FucT, for example by the methods described below.

[0188] Assays for changes in gene expression are well known in the art(see e.g., PCT Publication No. WO 96/34099, published Oct. 31, 1996,which is incorporated by reference herein in its entirety).

[0189] In particular, the assays may detect the presence of increased ordecreased expression of α1→2FucT gene or protein on the basis ofincreased or decreased mRNA expression (using, e.g., nucleic acidprobes), increased or decreased levels of related protein products(using, e.g., the antibodies disclosed herein), or increased ordecreased levels of expression of the catalytic product of the α1→2FucTgene (e.g. Fucosyl-GM₁).

[0190] 5.9.2. Gene Therapy

[0191] As α1→2FucT expression is often activated in cell transformation,e.g. small cell lung carcinoma, down-regulation of α1→2FucT expression,e.g. by anti-sense nucleic acids to α1→2FucT coding sequences, may beused to inhibit, suppress or treat cancer(see, supra, Section 5.7). Inone illustrative example, the anti-sense sequences are transduced inviral vectors, e.g. adenoviral or retroviral vectors.

[0192] 5.9.3. Preparative Synthesis of Fucosyl-Saccharide Compositions

[0193] The specificity of α1→2FucT of the invention lies in itsrecognition of the carbohydrate structure, Galβ1→3GalNAc, when found atthe terminus of a molecule. While the enzyme is utilized in vivo tocatalyze the addition of fucose in an α1→2 linkage to the terminalgalactose residue of the ganglioside GM₁, in vitro it is used tocatalyze the addition of fucose in an α1→2 linkage to the terminalgalactose residue of any molecule having a terminal Galβ1→3GalNAcmoiety. Such molecules include glycoproteins, glycolipids,glycolipoproteins and oligo- or poly-saccharides.

[0194] The α1→2FucT proteins of the invention may be used in any ofnumerous forms known in the art, e.g., as an isolated or purifiedprotein in solution, in a cellular fraction of a cell population thatexpresses the α1→2FucT proteins (see, supra, Section 5.2) orimmobilized, for example on a substrate or planar surface or inliposomes, micelles, microparticles, or microcapsules, etc.

[0195] According to one embodiment of the present invention, theα1→2FucT protein (or a catalytic derivative or analog thereof) can beused in the preparative synthesis of a molecule which comprises aFucα1→2Galβ1→3GalNAc moiety, said method comprising contacting isolatedor purified rat α1→2FucT of the invention with GDP-fucose and a moleculehaving a terminal Galβ1→3GalNAc moiety for a time sufficient to permitthe rat α1→2FucT to transfer the fucose to said molecule and recoveringa molecule which comprises Fucα1→2 Galβ1→3GalNAc. In one mode of thisembodiment, the molecule having a terminal Galβ1→3GalNAc moiety is aglycolipid, a glycoprotein, a glycolipoprotein or an oligo- orpolysaccharide. The oligo- or polysaccharide can be a free saccharide orcan be an saccharide immobilized, for example by means of a linkermoiety to a substrate or surface. A free saccharide having a Fucα1→2Galβ1→3GalNAc moiety can be obtained by cleavage of the linker moiety.

[0196] According to an alternative embodiment of the present invention,a cell fraction having catalytic activity of α1→2FucT protein (or acatalytic derivative or analog thereof) can be used in the preparativesynthesis of a molecule which comprises a Fucα1→2 Galβ1→3GalNAc moiety,said method comprising contacting a cell fraction having rat α1→2FucT ofthe invention with GDP-fucose and a molecule having a terminalGalβ1→3GalNAc moiety for a time sufficient to permit the rat α1→2FucT totransfer the fucose to said molecule and recovering a molecule whichcomprises Fucα1→2 Galβ1→3GalNAc. In one mode of this embodiment, themolecule having a terminal Galβ1→3GalNAc moiety is a glycolipid, aglycoprotein, a glycolipoprotein or an oligo- or polysaccharide. Theoligo- or polysaccharide can be a free saccharide or can be a saccharideimmobilized, for example by means of a linker moiety to a substrate orsurface. A free saccharide having a Fucα1→2Galβ1→3GalNAc moiety can beobtained by cleavage of the linker moiety.

[0197] According to a specific embodiment, the α1→2FucT (or a catalyticderivative or analog thereof) is used for the preparative synthesis offucosyl-GM₁. In one non-limiting example of this specific embodiment,fucosyl-GM₁ is prepared as follows: a reaction mixture composed of 25μmol of HEPES buffer, pH 7.2, 10 μmol of MnCl2, 500 μg CHAPSO, 0.5 mgGM₁ is contacted with a crude cell homogenate of COS-7 cells transientlytransfected with plasmid containing the rat α1→2fucosyltransferasecoding sequence. Progress of the reaction can be followed with time bywithdrawing aliquots of the reaction mixture and spotting it on an HPTLCplate. The plate is then developed in a solvent system composed ofCHCL₃:CH₃OH:H₂O, 60:40:9, containing 0.02% CaCl₂. Glycolipid bands areetermined by orcinol spray. Fucosyl-GM₁ is recovered.

[0198] 5.9.3.1. Uses of Saccharide Compositions Produced by α1→2FucT

[0199] The glycoproteins, glycolipids, glycolipoproteins or free oligo-or polysaccharides containing a Fucα1→2 Galβ1→3GalNAc moiety produced byα1→2FucT possess nutritional value, and as such may be used as foodadditives for e.g. infant formula or geriatric formula.

[0200] 5.9.3.2. Use of Fucosyl-GM₁ as an Immunosuppressive orImmunotherapeutic

[0201] Fucosyl-GM₁ is a cell surface antigen present on a variety oftumors. Thus, fucosyl-GM₁ can serve as a vaccine when presented to theimmune system of an individual with a tumor expressing this antigen bymethods known to those skilled in the art. In one embodiment,fucosyl-GM₁ prepared is injected directly into the bloodstream of theindividual, where it will elicit an immune response, resulting in theproduction of antibodies by B-cells against fucosyl-GM₁, whichantibodies will recognize cells of the tumor. In another embodiment,dendritic cells are extracted from an individual (e.g. by fluorescentactivated cell sorting (FACS) using an antibody against a cell surfaceantigen of dendritic cells as described in U.S. Pat. No. 5,876,917).Preferably, the dendritic cells are induced to proliferate in vitro(e.g. by the method of U.S. Pat. No. 5,851,756). The dendritic cells,whether having been induced to proliferate in vitro or not, are thenexposed to fucosyl-GM₁. These cells engulf the fucosyl-GM₁ antigen andpresent it on their cell surfaces. After re-introducing thefucosyl-GM₁-presenting cells into the patient, either into thebloodstream or locally at the site of the tumor, the cells willstimulate an immune response by activating T-cells, again resulting inthe production of anti-tumor antibodies and/or a cytotoxic cellularT-cell immune response against the tumor. Alternatively, the dendriticcells exposed to fucosyl-GM₁ can be used in vitro to stimulate T-cellsof the individual which T-cells can then be administered to the patientto afford a cellular immune response.

[0202] The present invention is further illustrated by the followingnon-limiting examples.

6. EXAMPLE Cloning and Expression of the Catalytic Domain if RatHepatoma GDP-Fucose:Gm₁ α1→2Fucosyltransferase

[0203] This example illustrates the cloning and expression of thecatalytic domain from rat hepatoma H35 cell GDP-fucose: GM₁α1→2fucosyltransferase, an enzyme which is activated during early stagesof chemical carcinogenesis in rat liver.

[0204] We have prepared primers based upon consensus sequences of highlyconserved regions of the α1→2FucT gene and, using an RT-PCR approach,amplified a product from H35 cell total RNA. These results haveindicated that H35 cells encode a novel enzyme, a portion of the 3′ endof which has previously been cloned from rat colonic adenocarcinoma PRObcells (Piau, J. -P., et al., 1994, Biochem. J. 300:623-626). Using thisinformation and additional primers from the more 5′ end of the gene, wehave cloned and expressed a 353 amino acid enzyme construct from H35cell total RNA with α1→2fucosyltransferase enzyme activity.

[0205] 6.1. Materials

[0206] Rat hepatoma H35 cells and simian COS-7 cells were obtained fromthe American Type Cell Collection (Manassas, Va.). RNAzol B total RNAisolation kit was obtained from Tel-Test, Inc. (Friendswood, Tex.).Plasmids pZErO-1 and pCR 2.1-TOPO were from Invitrogen (San Diego,Calif.) and pPROTA was received from Dr. Bruce Macher (San FranciscoState Univ., San Francisco, Calif.). Rabbit IgG-agarose beads andDEAE-dextran were obtained from Sigma (St. Louis, Mo.). PCR primers weremade on a Beckman Oligo 1000 synthesizer. GDP-[¹⁴C]fucose and[α-³⁵S]dATP were obtained from Dupont NEN (Boston, Mass.). Non-automatedDNA sequencing was done using the Sequenase Version 2.0 DNA sequencingkit from United States Biochemical Corp. (Cleveland, Ohio) or theSequiTherm EXCEL II DNA sequencing kit from Epicentre Technologies(Madison, Wis.). All other reagents were of the highest qualitycommercially available.

[0207] 6.2. Methods

[0208] 6.2.1. Cell Culture

[0209] Rat hepatoma H35 cells and simian COS-7 cells were grown intissue culture plates in Dulbecco's modified Eagle's medium (DME),supplemented with 10% fetal calf serum. The cells were harvested andpassed 1:4 every 5-6 days.

[0210] 6.2.2. RT-PCR Analysis of Rat Hepatoma H35 Cells:α1→2Fucosyltransferase

[0211] Total RNA was extracted from approximately 1×10⁷ rat hepatoma H35cells or from 300 mg of F344 whole liver tissue using the RNAzol Bmethod (Tel-test, Inc.). The isolated RNA, in 10 mM Tris buffer, pH 7.5,was initially amplified by RT-PCR using the following primers: primer I(forward), 5′-GGCCGCTTTGGGAACCAGATGG-3′ (22-mer) (SEQ ID NO:1); primerII (reverse), 5′-GGTTACACTGCGTGAGCAGCGC-3′ (22-mer)(SEQ ID NO:2). Theseprimers were based upon the consensus of portions of human, rabbit andrat intestine α1→2FT coding sequences which have substantial sequencehomology. The location of these primers in relation to DNA sequences ofother α1→2 FucT enzymes is illustrated in FIG. 1.

[0212] cDNA was made from (˜75 μg total) RNA using random hexamers asprimers for MuLV reverse transcriptase. Amplification was then conductedwith AmpliTaq DNA polymerase using 200 pM of each of the above primersin 35 cycles of 95° C. for 30 s, 58° C. for 30 s, and 72° C. for 1 minin a Coy thermocycler using a Gene Amp PCR kit (Perkin-Elmer,Branchburg, N.J.) to obtain a PCR product of approximately 0.6-kb.

[0213] Some DNA sequence was obtained using the Sequenase PCR productsequencing kit (USB/Amersham, Cleveland, Ohio) for direct sequencing ofPCR products using the dideoxy chain termination method (Sanger, F., etal., 1977, Proc. Natl. Acad. Sci. USA). The 0.6-kb rat PCR product wasalso cloned into the EcoRV site of pZErO plasmid (Invitrogen) andsequenced using the Sequenase version 2.0 sequencing kit (USB/Amersham)in order to determine the sequence near the 5′ and 3′ ends of theproduct.

[0214] Based upon sequencing results, which revealed 99% identitybetween 197 nucleotides at the 3′ end of the 0.6-kb PCR product and the5′ end of the rat α1→2FTB reported earlier (Piau, J. -P., et al., 1994,Biochem. J. 300:623-626), a second reverse primer was also made, whichis homologous with the 3′ end of the coding portion of the rat FTB genewith stop codon (shown in bold lettering below) and some 3′ untranslatedsequence: primer III (reverse), 5′-TTCCCATCAGAAGGCTCTTCCTGC-3′ (SEQ IDNO:3). A second, more upstream forward primer was made based upon rabbitRFT-III, which was found to be the most homologous gene on thenucleotide level to our rat PCR product. This 17-base-pair primerencompassed nucleotides 62-78 of rabbit RFT-III, within the regiondetermined to be near the end of the hydrophobic transmembrane domain ofthe enzyme. Although sequence homology between differing α1→2FucT genesis considerably reduced in this region compared to more 3′ sequences,this particular short sequence showed a reasonable degree of homology toaligned regions of rabbit RFT-II (nucleotides 71-87) and human Sec2(nucleotides 29-45) genes as well (see FIG. 1). This primer was asfollows:

[0215] primer IV(forward), 5′-CCGCCTCCACCATCTTC-3′ (SEQ ID NO:4). RT-PCRwas conducted on total rat H35 cell RNA as described above to obtain aPCR product of approximately 1.1 kb which was cloned into pCR 2. 1-TOPOvector and sequenced. A final forward primer was made reflectingexclusively the H35 α1→2FucT gene sequence and adaptors for cloning intothe pPROTA fusion protein expression vector: primer V (forward),5′-ATgaattcCCTCCAGCAGCGAATA-3′ (SEQ ID NO:5). An EcoRI site (shown inlower case above) and an additional C residue (bold) were included inthe forward primer for in frame cloning into pPROTA (Henion et al.,1994, Glycobiology 4:193-202).

[0216] 6.2.3. Construction of a Rat α1→2FucT Expression Vector andExpression of RT-PCR cDNA

[0217] RT-PCR was performed on rat H35 cell total RNA using primercombinations III (SEQ ID NO:3) and V (SEQ ID NO:5), as described above,to obtain a 1.077-kb PCR product, which was then subcloned into pCR2.1-TOPO. The insert was excised with EcoRI and subsequently cloned intothe EcoRI site of pPROTA plasmid for the production of the Protein A-IgGbinding domain/rat H35 cell α1→2FucT fusion protein (Henion, T. R., etal., 1994, Glycobiology 4:193-202). Correct orientation of the PCRinsert was established by HindIII StuI digestion and the resultantconstruct named CAT-RFT-pPROTA. CAT-RFT-pPROTA was transientlytransfected into COS-7 cells by the DEAE-dextran method (Ausubel, F. M.,et al., 1993, Current Protocols in Molecular Biology, Wiley, N.Y.).Secreted fusion protein was purified from the conditioned medium ofcells after 4-5 days on IgG-agarose beads as previously described(Holmes, E. H., et al., 1995, J. Biol. Chem. 270:8145-8151) for theassay of α1→2FucT expression.

[0218] 6.2.4. α1→2Fucosyltransferase Assays

[0219] α1→2Fucosyltransferase activity was determined in reactionmixtures containing 2.5 μmol of HEPES buffer, pH7.2, 30 μg of GM₁ganglioside or nLc₄, 250 μg phosphatidylglycerol, 1 μmol of MnCl₂, 0.5μmol of CDP-choline, 15 nmol of GDP-[¹⁴C]fucose (15,000 cpm/nmol), andpPROTA-expressed enzyme bound to IgG-agarose beads in a total volume of0.1 ml. The reaction mixtures were incubated for 2 h at 37° C.,terminated by the addition of 0.1 ml of CHCl₃:CH₃OH (2:1), and streakedonto a 4-cm-wide strip of Whatman 3 paper and developed with waterovernight. The papers were dried and the labeled product extracted fromthe origins with 2- to 5-ml washes of CHCl₃:CH₃OH:H₂O (10:5:1). Thecombined eluates were concentrated to dryness by an N₂ stream anddissolved in 20 μl of CHCl₃:CH₃OH (2:1). A 10-μl aliquot of each wasspotted onto a HP-TLC plate (Merck)and developed in a solvent systemcomposed of CHCl₃:CH₃OH:H₂O (60:40:9), 0.02% CaCl₂.2H₂O. The radioactiveproducts were located by autoradiography.

[0220] 6.3. Results

[0221] 6.3.1. RT-PCR Analysis of α1→2FucT Expression in Rat Hepatoma H35Cells

[0222] A survey of aligned nucleotide sequences for human and rabbitα1→2FucT enzyme genes indicates areas where very high sequence homologyexists between all forms. Portions of these aligned sequences are shownin FIG. 1. Two of these regions were selected for PCR primer design andinitial RT-PCR amplification of H35 cell total RNA. The location ofthese regions (designated primers I and II; SEQ ID NO.s:1 and 2),corresponding to nucleotides 220 to 241 and 838 to 859 of the rabbitRFT-III for comparison, are also shown in FIG. 1.

[0223] A single PCR product slightly over 0.6-kb in size was obtainedusing primers (SEQ ID NO:1) and II (SEQ ID NO:2) (FIG. 2, lane 1), whichcorresponds to the expected fragment size based upon location of theseprimer regions in the gene. Sequencing on both strands revealed a run of597 unambiguous nucleotides between the two primer sequences, which werecompared to rabbit and rat α1→2FTs. Up to 84% homology in nucleotidesequence was detected between this rat PCR product and the rabbitgene(s). The last 197 nucleotides at the 3′ of the PCR product werefound to have 99% identity with the 5′ end of the rat α1→2FTB fragmentreported earlier (Piau, J. -P., et al., 1994, Biochem. J. 300:623-626).The difference (GTG) was detected at the codon for amino acid 50 encodedby the rat FTB fragment (GGT) and was confirmed on two PCR clones with 3different primers. This represents an amino acid change of glycine inFTB to valine in the H35 cell α1→2FucT at that site. No RT-PCR productwas obtained from H35 cell total RNA corresponding to rat FTA (Piau, J.-P., et al., 1994, Biochem. J. 300:623-626) using rat FTA primers(results not shown).

[0224] To verify that sequences from the rat FTB fragment constitutedthe 3′ region of the H35 cell α1→2FucT gene, a second RT-PCR experimentwas performed using primers I (forward) (SEQ ID NO:1) and III (reverse)(SEQ ID NO:3) (see, supra, Sections 6.1 and 6.2). These primers reflectthe start site used in generating the first PCR product through the endof the gene based upon the FTB sequence. As shown in FIG. 2, lane 2, aproduct, approximately 0.9 kb in size, was obtained from rat H35 totalRNA. This PCR product was sequenced and confirmed that rat FTB mostprobably corresponds to the 3′ portion of this gene.

[0225] In general, mammalian membrane-bound glycosyltransferases arecomposed of a short intracellular N-terminal domain, a transmembranedomain, and an extracellular stem region and C-terminal catalyticdomain. The stem region corresponds to portions of the extracellulardomain which can be removed and are not required for catalytic activity.Generally, most sequence homology among α1→2FucT enzymes occur in thecatalytic domain with much lower homology found in DNA sequencescorresponding to the more N-terminal portion of the protein. To obtainan RT-PCR product from H35 cell total RNA containing sequences for asmuch of the N-terminal of the protein as possible to ensure an activeenzyme would later be expressed, a forward primer (primer IV; SEQ IDNO:4) 1 corresponding to portions of the transmembrane domain of rabbitRFT-III (nucleotides 62 to 78) where reasonable sequence homology existsbetween enzymes was used in combination with primer III (SEQ ID NO:3).The results (not shown) indicated that a PCR product of approximately1.1 kb was generated. Sequencing of this product confirmed that itcontained the same sequence obtained in the earlier RT-PCR experimentsand included an additional 181 nucleotides of rat H35 cell α1→2FucTsequence at the 5′ end.

[0226] To obtain a cDNA containing only confirmed rat α1→2FucT sequencesfor insertion into the EcoRI site of the pPROTA expression vector, aforward primer (primer V; SEQ ID NO:5) was used in combination withprimer III (SEQ ID NO:3) in an RT-PCR experiment. Primer V (SEQ ID NO:5)corresponded to the most 5′ end of the confirmed rat sequence andcontained an adaptor for EcoRI cloning and an extra C residue forin-frame cloning into the pPROTA vector. A product of approximately 1.1kb (1068 nucleotides of confirmed rat H35 cell α1→2FucT sequence) wasamplified from rat H35 cell total RNA using primers V and III (FIG. 2,lane 3). This product represents the majority of the rat H35 cellα1→2FucT, but is missing the start of the coding sequence encodingintracellular and transmembrane domains of the protein. This PCR productwas fully sequenced (FIG. 3A) and determined to encode the α1→2FucTassociated with malignant transformation in rat liver cells.

[0227] Sequence analysis using the BLAST algorithm (Altschul et al.,1990, J. Mol. Biol. 215(3):403-10) determined that the observed sequenceis highly homologous to the sequences of all presently known α1→2FucTcoding sequences from human, rabbit and rat. It is also virtuallyidentical to the 5′ 480 nucleotides of the fragment from rat FTBisolated by Piau et al. (1994, Biochem. J. 300:623-626), which encodes apolypeptide comprising approximately half of the α1→2FucT catalyticdomain and possessing no catalytic activity. The sequence shown in FIG.3A codes for 353 amino acids and contains four potential N-linkedglycosylation sites. Table I shows the comparative extent of nucleotideand deduced amino acid sequence from all known enzyme forms. Asindicated, high homology was detected between the amino acid sequencesof the human Sec2 enzyme and the rat H35 cell α1→2FucT enzyme at 77%.FIG. 3B shows an aligned deduced amino acid sequence comparison betweenthese two enzymes. Rabbit RFT-II and RFT-III enzymes also show a highdegree of homology at 71% and 68%, respectively, and rat FTA fragment at70%. There is far less sequence homology between the rat H35 cellα1→2FucT and the human H and the rabbit RFT-I enzymes. Thus, this newrat enzyme appears to be more closely related to the secretor enzymethan the H enzyme. This is consistent with published results (Larsen, R.D., et al., 1990, Proc. Natl. Acad. Sci. USA 87:6674-6678; Kelly, R. J.,et al., 1995, J. Biol. Chem. 270:4640-4649; Hitoshi, S., et al., 1995,J. Biol. Chem. 270:8844-8850; i Hitoshi, S., et al., 1996, J. Biol.Chem. 271:16975-16981) which show a proportionally higher specificityfor GM₁ acceptors compared to lacto- or neolacto-series acceptors forsecretor enzyme-like forms compared to the H enzyme. TABLE I Comparisonof Percent Homology of the Catalytic Domain of Rat Hepatoma H35 Cell α1→ 2FucT with Other Cloned α1 → 2FucT Enzyme Sequences % Homology¹ basedon Deduced Amino Acid Enzyme Nucleotide Sequence Sequence Human H 62 58Human Sec2 73 77 Human Sec1 69 66 Rabbit FT-I 64 59 Rabbit FT-II 71 71Rabbit FT-III 75 68 Rat FTA 69 70 Rat FTB 99 99

[0228] 6.3.2. Analysis of pPROTA-Expressed H35 Cell α1→2FucT Activity

[0229] Expression of CAT-RFT-pPROTA results in the production of afusion protein composed of the protein A-IgG-binding domain and theα1→2FucT sequence (SEQ ID NO:10) shown in FIG. 3A. The expressed proteinis conveniently isolated by binding to IgG-agarose beads which can bedirectly assayed for enzyme activity. As shown in FIG. 4, lane 1, theexpressed H35 cell α1→2FucT was found to transfer fucose to GM₁. Nodetectable transfer was observed to the neolacto-series acceptornLcOse₄Cer (lane 3), whose carbohydrate moiety is characterized by aterminal Galβ1→4GlcNAcβ1 saccharide. Further, no transfer to GM₁ wasobserved with beaded enzyme obtained after inserting the H35 cellα1→2FucT cDNA into pPROTA in the reverse orientation (lane 2).

[0230] 6.4. Discussion

[0231] Aligned sequences of human and rabbit α1→2FucT's demonstrateconsiderable homology in regions corresponding to the catalytic domainof the enzyme. According to the present invention, an RT-PCR cloningstrategy utilizing primers corresponding to consensus sequences betweenthese genes was successful in amplifying the appropriate coding sequencefrom rat H35 cell total RNA. The results demonstrate that this approachprovided a significant portion of the H35 cell α1→2FucT sequence. Theinitial PCR sequence which was illustrated in FIG. 3A overlapped withthat from the previously published rat FTB fragment (Piau, J. -P., etal., 1994, Biochem. J. 300:623-626). The rat FTB sequence, when placedin tandem with our upstream sequence, yielded a coding sequence for 292amino acids and a stop codon. Subsequent use of a primer encompassingthe sequence surrounding this stop codon, as well as another encoding aportion of the transmembrane domain of rabbit RFT-III, yielded a cDNAencoding the extracellular portion of the rat H35 cell α1→2FucT. ThecDNA corresponding to confirmed rat α1→2FucT sequences when expressed inthe pPROTA vector yielded a protein A-IgG-binding domain fusion proteinwith GM₁-specific cα1→2FucT activity.

[0232] The observed cDNA sequence of the H35 cell α1→2FucT was found tobe distinct yet highly homologous to relevant portions of the genes fromother species (Larsen, R. D., et al., 1990, Proc. Natl. Acad. Sci. USA87:6674-6678; Kelly, R. J., et al., 1995, J. Biol. Chem. 270:4640-4649;Hitoshi, S., et al., 1995, J. Biol. Chem. 270:8844-8850; Hitoshi, S., etal., 1996, J. Biol. Chem. 271:16975-16981; Piau, J. -P., et al., 1994,Biochem. J. 300:623-626). As indicated above, the H35 cell α1→2FucT cDNAobtained is missing 5′ regions of the gene encoding the intracellularand transmembrane domains of the enzyme, corresponding to an estimated15 to 30 amino acids from the N-terminal of the protein based uponsequence alignments with cloned full length proteins. In general, thisregion has a lower degree of sequence homology in comparison with otherα1→2FucT's. Thus, the degree of homology contained within only theextracellular domain may be slightly higher than if the entire codingsequences are compared.

[0233] The results indicate that repeated RT-PCR experiments withseveral primers provided cDNA products with clear, unambiguous, andidentical sequences. There was no evidence suggesting multiple PCRproducts were generated with any primer combination used. In particular,no sequence corresponding to the rat FTA gene (Piau, J. -P., et al.,1994, Biochem. J. 300:623-626) was obtained, even when primers specificfor FTA were used. Thus, rat hepatoma H3 5 cells most probably expressonly a single α1→2FucT enzyme, one with very high specificity forganglio-series acceptors.

7. EXAMPLE Cloning and Expression of Full Length GDP-Fucose:GM₁ α1→2FucT

[0234] 7.1. Cloning

[0235] We have cloned the entire coding region of the rat α1→2FucT gene.Based upon information obtained from a 2984 bp Rattus norvegicus FTBmRNA sequence found in GenBank databases (Koda, Y., Submitted to theDDBJ/EMBL/GenBank databases, 1997, Accession #AB006138), a forwardprimer was designed from the putative start of translation, determinedby the rules of Kozak (Kozak, M., 1992, Ann. Rev. Cell Biol. 8:197-225).This mRNA was found to contain 213 nucleotides of upstream untranslatedsequence and over 1580 bp of 3′ untranslated sequence. As it is reportedin the GenBank database, this particular sequence has an error at aminoacid 354 (A-H) immediately followed by a nonsense mutation (TAA) forpremature termination and therefore, would not express an active enzyme.In addition to the amino acid 354 (AH) change, this sequence (asreported) is missing 26 crucial amino acids at the C terminus. Theseare:

[0236] LTPACPRSHFHLKAKGVTCYVAGRAF (amino acids 355-380 of SEQ ID NO:8).However, based on correct sequence information at the 5′ end, a newforward primer was designed:

[0237] Primer VI: 5′GCCATGGCCAGCGCCCAGGTTCCT 3′ (SEQ ID NO:6). Thisprimer was used in conjunction with Primer III (SEQ ID NO:3), describedabove, to RT-PCR the entire 1140 bp α1→2FucT coding region (results notshown). This PCR product was sequenced (FIG. 5) and determined to be thenucleotide sequence encoding the α1→2FucT associated with malignanttransformation in rat liver cells. Table II shows the comparative extentof nucleotide and deduced amino acid sequence from all known enzymeforms. Full length α1→2FucT cDNA was then cloned into pcDNA 3 vector(Invitrogen) in both the positive (FL-RFT-pcDNA3) and negative(FL-RFT(−)-pcDNA3) orientations for later α1→2FucT enzyme assays. TABLEII Comparison of Percent Homology of the Full Length Rat Hepatoma H35Cell α1 → 2FucT with Other Cloned α1 → 2FucT Enzyme Sequences %Homology¹ based on Deduced Amino Acid Enzyme Nucleotide SequenceSequence Human H 62 55 Human Sec2 74 77 Human Sec1 70 63 Rabbit FT-I 6456 Rabbit FT-II 71 67 Rabbit FT-III 75 65 Rat FTA 69 70 Rat FTB 99 99

[0238] 7.2. Analysis of Expressed Full Length α1→2FucT Activity

[0239] Expression of full length α1→2FucT cDNA in transfected cellsresults in membrane-bound enzyme. COS-7 cells were transientlytransfected by the DEAE-dextran method (Ausubel, F. M., et al., CurrentProtocols in Molecular Biology, Wiley, N.Y.) with either FL-RFT-pcDNA3or FL-RFT(−)-pcDNA3. Four to five days later, the cells were harvested,sonicated in HEPES, glycerol, DTE buffer and assayed for enzyme activityas described previously (Sherwood, A. L., et al., 1998, Arch. Biochem.Biophys. 355:215-221).

[0240] As shown in FIG. 6, the expressed recombinant full length enzymetransfers fucose to GM₁ with high efficiency; much higher than thepPROTA-expressed truncated enzyme. Comparable transfer was observed inthe presence or absence of CHAPSO detergent (100 μg); (lanes D and A,respectively). Somewhat less transfer to GM₁ was observed in thepresence of 250 μg of phosphatidylglycerol (lane B) and significantlyless transfer was observed in the presence of both phosphatiylglycerol(250 μg) and G3634A detergent (100 μg) (Lane C). No fucose transfer wasobserved under any of these conditions in homogenates from COS-7 cellstransfected with FL-RFT(−)-pcDNA3 (results not shown).

8. EXAMPLE RT-PCR of α1→2FucT in Rat Liver After Administration of aCarcinogen

[0241] Expression of α1→2FucT in F344 rat liver before and afteradministration of the carcinogen 0.03% N-2-acetylaminofluorene (AAF) inthe diet (Holmes, E. H., 1990, Carcinogenesis 11:89-94) was tested byRT-PCR of total RNA using primers I and II and compared to the resultswith H35 cell total RNA. Total RNA was extracted from approximately 200mg of normal, healthy Fisher 344 rat liver tissue and 200 mg of livertissue from rats fed a diet containing 0.03% AAF for >3 weeks using theRNAzol B method (Tel-test, Inc., Friendswood, Tex.). RT-PCR wasconducted as described (Sherwood, A. L., et al., 1998, Arch. Biochem.Biophys. 355:215-221), with 200 pM of primers I and II which have beenfound to reproducibly yield a single PCR product of approximately0.6-kb. This represents a portion of the GDP-fucose:GM₁ specificα1→2FucT present in rat hepatoma H35 cells. Results are shown in FIG. 7.

[0242] As shown in FIG. 7, an approximately 0.6-kb product correspondingto that derived from H35 cell total RNA (lane 1) was obtained with totalRNA derived from liver after 0.03% AAF feeding (lane 3). No PCR productwas obtained in the same experiment from total RNA isolated from normalF344 liver (lane 2). The AAF-fed rat liver sample chosen for this studywas one of several which displayed a moderate level of α1→2FucT enzymeactivity following a feeding regimen of AAF carcinogen. An identical PCRproduct was also obtained in a later experiment using a second AAF-fedrat liver sample, which had previously been found to display low tomoderate α1→2FucT enzyme activity (results not shown). No α1→2FucTenzyme activity has ever been detected in normal liver tissue from ratsfed a standard diet lacking AAF. The results presented in FIG. 7 clearlydemonstrate that mRNA encoding the α1→2FucT gene is not expressed innormal F344 rat liver tissue but is present in liver tissue afteradministration of 0.03% AAF. The observation that both enzyme activityand mRNA specific for α1→2FucT is present after only three or more weeksof exposure to AAF confirms that this enzyme is induced in the earlystages of chemical carcinogenesis in rat liver.

[0243] The observation of the induction of synthesis of this enzymeduring very early stages of chemical carcinogenesis suggests that it isan interesting marker for studying this process in vivo. Resultspresented confirm that mRNA encoding the α1→2FucT gene is not expressedin normal F344 rat liver tissue but is present in liver tissue afteradministration of 0.03% N-2-acetylaminofluorene.

9. EXAMPLE Inhibition of α1→2FucT Activity by Antisense Treatment

[0244] The ability of antisense α1→2FucT nucleotides to inhibit α1→2FucTactivity was assessed in COS-7 cells in which a constant “dose” (1 μg)of FL-RFT-pcDNA3 sense cDNA was transiently transfected with increasing“doses” (1, 2, 3 and 5 fig) of FL-RFT(−)-pcDNA3 antisense cDNA andvarying amounts of pcDNA3 vector (no insert) in each case to maintainequi-molar ratios of total plasmid transfected into cells under eachcondition. Four to five days later, COS-7 cells were harvested,sonicated in HEPES, glycerol, DTE buffer and assayed for α1→2FucTactivity as previously described (Sherwood, A. L., et al., 1998, Arch.Biochem. Biophys. 355:215-221). A progressive decrease in enzymeactivity was observed with increasing concentrations of antisenseα1→2FucT cDNA (FIG. 8). We chose to initially test this system in COS-7cells because we have had consistently excellent (and rapid) resultsexpressing constructs of various human α1→3fucosyltransferase genes (aswell as rat α1→2FucT constructs) in this line. We currently haveFL-RFT(−)-pcDNA3 stably transfected H35 hepatoma cells undergoingselection in G418 medium. Our results demonstrate a highly effectiveantisense treatment system for the down-regulation of rat α1→2FucT.

10. EXAMPLE Preparative in vitro Biosynthesis of Fucosyl-GM₁ UtilizingRecombinant Rat α1→2Fucosyltransferase

[0245] Preparative biosynthesis of fucosyl-GM₁ was conducted in reactionmixtures composed of 25 μmol of HEPES buffer, pH 7.2, 10 Smog of MnCl2,500 μg CHAPSO, 0.5 mg GM₁, and 2 mg crude cell homogenate of COS-7 cellstransiently transfected with plasmid containing the entire ratα1→2fucosyltransferase coding sequence in a total volume of 0.5 ml.Progress of the reaction was followed with time by withdrawing 2 ill ofthe reaction mixture and spotting it on an HPTLC plate. The plate wasdeveloped in a solvent system composed of CHCL₃:CH₃OH:H₂O, 60:40:9,containing 0.02% CaCl₂. Glycolipid bands were determined by orcinolspray (FIG. 9).

[0246] The results demonstrate the appearance of increasing amounts of aslower migrating band corresponding to fucosyl-GM₁ from transfer offucose in the α1→2-linkage to the added GM₁ acceptor with time. Theenzyme is very active yielding almost complete conversion to fucosyl-GM₁after 24 to 48 hours. This preparative biosynthesis can be scaledappropriately to provide any amount of fucosyl-GM₁ product needed and isadvantageously useful for commercial scale production of fucosyl-GM₁.

11. Deposit of Microorganisms

[0247] The following microorganisms were deposited with the AmericanType Culture Collection (ATCC), 10801 University Blvd., Manassas, Va. onApr. 22, 1999 and have been assigned accession numbers as indicated.Microorganism Accession Number CAT-RFT-pPROTA in E. coli INVα —FL-RFT-pcDNA3 in E. coli DH5α —

[0248] The present invention is not to be limited in scope by thespecific embodiments described herein. Indeed, various modifications ofthe invention in addition to those described herein will become apparentto those skilled in the art from the foregoing description andaccompanying drawings. Such modifications are intended to fall withinthe scope of the appended claims.

[0249] Various references are cited herein above, including patentapplications, patents, and publications, the disclosures of which arehereby incorporated by reference in their entireties.

1 29 1 22 DNA Artificial Sequence Description of Artificial Sequenceprimer 1 ggccgctttg ggaaccagat gg 22 2 22 DNA Artificial SequenceDescription of Artificial Sequence primer 2 ggttacactg cgtgagcagc gc 223 24 DNA Artificial Sequence Description of Artificial Sequence primer 3ttcccatcag aaggctcttc ctgc 24 4 17 DNA Artificial Sequence Descriptionof Artificial Sequence primer 4 ccgcctccac catcttc 17 5 24 DNAArtificial Sequence Description of Artificial Sequence primer 5atgaattccc tccagcagcg aata 24 6 24 DNA Artificial Sequence Descriptionof Artificial Sequence primer 6 gccatggcca gcgcccaggt tcct 24 7 1149 DNARattus norvegicus CDS (1)..(1143) 7 atg gcc agc gcc cag gtt cct ttc tccttt cct ctg gcc cac ttc ctc 48 Met Ala Ser Ala Gln Val Pro Phe Ser PhePro Leu Ala His Phe Leu 1 5 10 15 atc ttt gtc ttc gtg act tcc acc atcatc cac ctc cag cag cga ata 96 Ile Phe Val Phe Val Thr Ser Thr Ile IleHis Leu Gln Gln Arg Ile 20 25 30 gtg aag ctc caa ccc ctg tca gag aag gaatta ccg atg acg act caa 144 Val Lys Leu Gln Pro Leu Ser Glu Lys Glu LeuPro Met Thr Thr Gln 35 40 45 atg tcc tcg gga aac aca gaa agc cca gag atgcga cgg gac agc gag 192 Met Ser Ser Gly Asn Thr Glu Ser Pro Glu Met ArgArg Asp Ser Glu 50 55 60 cag cat ggg aat gga gag ctg cgg ggc atg ttc acgatc aat tcc att 240 Gln His Gly Asn Gly Glu Leu Arg Gly Met Phe Thr IleAsn Ser Ile 65 70 75 80 ggc cgg ctg ggg aac cag atg ggc gaa tac gcc acactc ttt gca ctg 288 Gly Arg Leu Gly Asn Gln Met Gly Glu Tyr Ala Thr LeuPhe Ala Leu 85 90 95 gcc agg atg aac gga cgg ctt gcg ttc atc ccc gca tccatg cac aac 336 Ala Arg Met Asn Gly Arg Leu Ala Phe Ile Pro Ala Ser MetHis Asn 100 105 110 gct cta gcg ccc atc ttc agg atc agc ctc ccg gtg ttacac agc gac 384 Ala Leu Ala Pro Ile Phe Arg Ile Ser Leu Pro Val Leu HisSer Asp 115 120 125 acg gcc aaa aag atc cca tgg cag aat tac cat ctc aacgac tgg atg 432 Thr Ala Lys Lys Ile Pro Trp Gln Asn Tyr His Leu Asn AspTrp Met 130 135 140 gag gag cgt tac cgc cac att ccg gga cac ttt gtg cgcttc acg gga 480 Glu Glu Arg Tyr Arg His Ile Pro Gly His Phe Val Arg PheThr Gly 145 150 155 160 tac ccg tgc tcc tgg acc ttc tac cac cac ctg cgccca gag atc ctg 528 Tyr Pro Cys Ser Trp Thr Phe Tyr His His Leu Arg ProGlu Ile Leu 165 170 175 aag gag ttc acc ctg cat gac cac gtg cgg gag gaggcc cag gcc ttc 576 Lys Glu Phe Thr Leu His Asp His Val Arg Glu Glu AlaGln Ala Phe 180 185 190 ctg cgt ggt ctg cgg gtg aat ggg agc cag ccg agtact ttt gtg ggt 624 Leu Arg Gly Leu Arg Val Asn Gly Ser Gln Pro Ser ThrPhe Val Gly 195 200 205 gtc cat gtg cgc cga ggg gac tat gtg cat gtc atgcct aat gtg tgg 672 Val His Val Arg Arg Gly Asp Tyr Val His Val Met ProAsn Val Trp 210 215 220 aag ggc gtg gtg gct gac cgg ggt tac ctg gaa aaggcc ctg gat atg 720 Lys Gly Val Val Ala Asp Arg Gly Tyr Leu Glu Lys AlaLeu Asp Met 225 230 235 240 ttc cgg gca cgc tat tca tct cca gtc ttc gtggtt aca agc aac ggt 768 Phe Arg Ala Arg Tyr Ser Ser Pro Val Phe Val ValThr Ser Asn Gly 245 250 255 atg gcc tgg tgc cgg gag aac att aat gct tcccga gga gac gtg gtg 816 Met Ala Trp Cys Arg Glu Asn Ile Asn Ala Ser ArgGly Asp Val Val 260 265 270 ttc gcg ggc aat ggt att gag ggg tcg cca gccaag gac ttc gcg ctg 864 Phe Ala Gly Asn Gly Ile Glu Gly Ser Pro Ala LysAsp Phe Ala Leu 275 280 285 ctc acc cag tgc aac cac acc atc atg act attggg acc ttt ggg att 912 Leu Thr Gln Cys Asn His Thr Ile Met Thr Ile GlyThr Phe Gly Ile 290 295 300 tgg gct gcc tac ctg gca ggt ggt gat acc atctac tta gcc aac tac 960 Trp Ala Ala Tyr Leu Ala Gly Gly Asp Thr Ile TyrLeu Ala Asn Tyr 305 310 315 320 acc ctt ccg gat tct ccg ttc ctc aaa gtcttt aag cca gag gca gcc 1008 Thr Leu Pro Asp Ser Pro Phe Leu Lys Val PheLys Pro Glu Ala Ala 325 330 335 ttc cta ccc gaa tgg gtg ggc atc cct gccgat ctg tcc cca ctc ctt 1056 Phe Leu Pro Glu Trp Val Gly Ile Pro Ala AspLeu Ser Pro Leu Leu 340 345 350 aag gca tta aca cca gcc tgt cct cgg tcccac ttc cac ctc aag gca 1104 Lys Ala Leu Thr Pro Ala Cys Pro Arg Ser HisPhe His Leu Lys Ala 355 360 365 aaa gga gtc act tgt tac gtc gca gga agagcc ttc tga tgggaa 1149 Lys Gly Val Thr Cys Tyr Val Ala Gly Arg Ala Phe370 375 380 8 380 PRT Rattus norvegicus 8 Met Ala Ser Ala Gln Val ProPhe Ser Phe Pro Leu Ala His Phe Leu 1 5 10 15 Ile Phe Val Phe Val ThrSer Thr Ile Ile His Leu Gln Gln Arg Ile 20 25 30 Val Lys Leu Gln Pro LeuSer Glu Lys Glu Leu Pro Met Thr Thr Gln 35 40 45 Met Ser Ser Gly Asn ThrGlu Ser Pro Glu Met Arg Arg Asp Ser Glu 50 55 60 Gln His Gly Asn Gly GluLeu Arg Gly Met Phe Thr Ile Asn Ser Ile 65 70 75 80 Gly Arg Leu Gly AsnGln Met Gly Glu Tyr Ala Thr Leu Phe Ala Leu 85 90 95 Ala Arg Met Asn GlyArg Leu Ala Phe Ile Pro Ala Ser Met His Asn 100 105 110 Ala Leu Ala ProIle Phe Arg Ile Ser Leu Pro Val Leu His Ser Asp 115 120 125 Thr Ala LysLys Ile Pro Trp Gln Asn Tyr His Leu Asn Asp Trp Met 130 135 140 Glu GluArg Tyr Arg His Ile Pro Gly His Phe Val Arg Phe Thr Gly 145 150 155 160Tyr Pro Cys Ser Trp Thr Phe Tyr His His Leu Arg Pro Glu Ile Leu 165 170175 Lys Glu Phe Thr Leu His Asp His Val Arg Glu Glu Ala Gln Ala Phe 180185 190 Leu Arg Gly Leu Arg Val Asn Gly Ser Gln Pro Ser Thr Phe Val Gly195 200 205 Val His Val Arg Arg Gly Asp Tyr Val His Val Met Pro Asn ValTrp 210 215 220 Lys Gly Val Val Ala Asp Arg Gly Tyr Leu Glu Lys Ala LeuAsp Met 225 230 235 240 Phe Arg Ala Arg Tyr Ser Ser Pro Val Phe Val ValThr Ser Asn Gly 245 250 255 Met Ala Trp Cys Arg Glu Asn Ile Asn Ala SerArg Gly Asp Val Val 260 265 270 Phe Ala Gly Asn Gly Ile Glu Gly Ser ProAla Lys Asp Phe Ala Leu 275 280 285 Leu Thr Gln Cys Asn His Thr Ile MetThr Ile Gly Thr Phe Gly Ile 290 295 300 Trp Ala Ala Tyr Leu Ala Gly GlyAsp Thr Ile Tyr Leu Ala Asn Tyr 305 310 315 320 Thr Leu Pro Asp Ser ProPhe Leu Lys Val Phe Lys Pro Glu Ala Ala 325 330 335 Phe Leu Pro Glu TrpVal Gly Ile Pro Ala Asp Leu Ser Pro Leu Leu 340 345 350 Lys Ala Leu ThrPro Ala Cys Pro Arg Ser His Phe His Leu Lys Ala 355 360 365 Lys Gly ValThr Cys Tyr Val Ala Gly Arg Ala Phe 370 375 380 9 1068 DNA Rattusnorvegicus CDS (1)..(1062) 9 ctc cag cag cga ata gtg aag ctc caa ccc ctgtca gag aag gaa tta 48 Leu Gln Gln Arg Ile Val Lys Leu Gln Pro Leu SerGlu Lys Glu Leu 1 5 10 15 ccg atg acg act caa atg tcc tcg gga aac acagaa agc cca gag atg 96 Pro Met Thr Thr Gln Met Ser Ser Gly Asn Thr GluSer Pro Glu Met 20 25 30 cga cgg gac agc gag cag cat ggg aat gga gag ctgcgg ggc atg ttc 144 Arg Arg Asp Ser Glu Gln His Gly Asn Gly Glu Leu ArgGly Met Phe 35 40 45 acg atc aat tcc att ggc cgg ctg ggg aac cag atg ggcgaa tac gcc 192 Thr Ile Asn Ser Ile Gly Arg Leu Gly Asn Gln Met Gly GluTyr Ala 50 55 60 aca ctc ttt gca ctg gcc agg atg aac gga cgg ctt gcg ttcatc ccc 240 Thr Leu Phe Ala Leu Ala Arg Met Asn Gly Arg Leu Ala Phe IlePro 65 70 75 80 gca tcc atg cac aac gct cta gcg ccc atc ttc agg atc agcctc ccg 288 Ala Ser Met His Asn Ala Leu Ala Pro Ile Phe Arg Ile Ser LeuPro 85 90 95 gtg tta cac agc gac acg gcc aaa aag atc cca tgg cag aat taccat 336 Val Leu His Ser Asp Thr Ala Lys Lys Ile Pro Trp Gln Asn Tyr His100 105 110 ctc aac gac tgg atg gag gag cgt tac cgc cac att ccg gga cacttt 384 Leu Asn Asp Trp Met Glu Glu Arg Tyr Arg His Ile Pro Gly His Phe115 120 125 gtg cgc ttc acg gga tac ccg tgc tcc tgg acc ttc tac cac cacctg 432 Val Arg Phe Thr Gly Tyr Pro Cys Ser Trp Thr Phe Tyr His His Leu130 135 140 cgc cca gag atc ctg aag gag ttc acc ctg cat gac cac gtg cgggag 480 Arg Pro Glu Ile Leu Lys Glu Phe Thr Leu His Asp His Val Arg Glu145 150 155 160 gag gcc cag gcc ttc ctg cgt ggt ctg cgg gtg aat ggg agccag ccg 528 Glu Ala Gln Ala Phe Leu Arg Gly Leu Arg Val Asn Gly Ser GlnPro 165 170 175 agt act ttt gtg ggt gtc cat gtg cgc cga ggg gac tat gtgcat gtc 576 Ser Thr Phe Val Gly Val His Val Arg Arg Gly Asp Tyr Val HisVal 180 185 190 atg cct aat gtg tgg aag ggc gtg gtg gct gac cgg ggt tacctg gaa 624 Met Pro Asn Val Trp Lys Gly Val Val Ala Asp Arg Gly Tyr LeuGlu 195 200 205 aag gcc ctg gat atg ttc cgg gca cgc tat tca tct cca gtcttc gtg 672 Lys Ala Leu Asp Met Phe Arg Ala Arg Tyr Ser Ser Pro Val PheVal 210 215 220 gtt aca agc aac ggt atg gcc tgg tgc cgg gag aac att aatgct tcc 720 Val Thr Ser Asn Gly Met Ala Trp Cys Arg Glu Asn Ile Asn AlaSer 225 230 235 240 cga gga gac gtg gtg ttc gcg ggc aat ggt att gag gggtcg cca gcc 768 Arg Gly Asp Val Val Phe Ala Gly Asn Gly Ile Glu Gly SerPro Ala 245 250 255 aag gac ttc gcg ctg ctc acc cag tgc aac cac acc atcatg act att 816 Lys Asp Phe Ala Leu Leu Thr Gln Cys Asn His Thr Ile MetThr Ile 260 265 270 ggg acc ttt ggg att tgg gct gcc tac ctg gca ggt ggtgat acc atc 864 Gly Thr Phe Gly Ile Trp Ala Ala Tyr Leu Ala Gly Gly AspThr Ile 275 280 285 tac tta gcc aac tac acc ctt ccg gat tct ccg ttc ctcaaa gtc ttt 912 Tyr Leu Ala Asn Tyr Thr Leu Pro Asp Ser Pro Phe Leu LysVal Phe 290 295 300 aag cca gag gca gcc ttc cta ccc gaa tgg gtg ggc atccct gcc gat 960 Lys Pro Glu Ala Ala Phe Leu Pro Glu Trp Val Gly Ile ProAla Asp 305 310 315 320 ctg tcc cca ctc ctt aag gca tta aca cca gcc tgtcct cgg tcc cac 1008 Leu Ser Pro Leu Leu Lys Ala Leu Thr Pro Ala Cys ProArg Ser His 325 330 335 ttc cac ctc aag gca aaa gga gtc act tgt tac gtcgca gga aga gcc 1056 Phe His Leu Lys Ala Lys Gly Val Thr Cys Tyr Val AlaGly Arg Ala 340 345 350 ttc tga tgggaa 1068 Phe 10 353 PRT Rattusnorvegicus 10 Leu Gln Gln Arg Ile Val Lys Leu Gln Pro Leu Ser Glu LysGlu Leu 1 5 10 15 Pro Met Thr Thr Gln Met Ser Ser Gly Asn Thr Glu SerPro Glu Met 20 25 30 Arg Arg Asp Ser Glu Gln His Gly Asn Gly Glu Leu ArgGly Met Phe 35 40 45 Thr Ile Asn Ser Ile Gly Arg Leu Gly Asn Gln Met GlyGlu Tyr Ala 50 55 60 Thr Leu Phe Ala Leu Ala Arg Met Asn Gly Arg Leu AlaPhe Ile Pro 65 70 75 80 Ala Ser Met His Asn Ala Leu Ala Pro Ile Phe ArgIle Ser Leu Pro 85 90 95 Val Leu His Ser Asp Thr Ala Lys Lys Ile Pro TrpGln Asn Tyr His 100 105 110 Leu Asn Asp Trp Met Glu Glu Arg Tyr Arg HisIle Pro Gly His Phe 115 120 125 Val Arg Phe Thr Gly Tyr Pro Cys Ser TrpThr Phe Tyr His His Leu 130 135 140 Arg Pro Glu Ile Leu Lys Glu Phe ThrLeu His Asp His Val Arg Glu 145 150 155 160 Glu Ala Gln Ala Phe Leu ArgGly Leu Arg Val Asn Gly Ser Gln Pro 165 170 175 Ser Thr Phe Val Gly ValHis Val Arg Arg Gly Asp Tyr Val His Val 180 185 190 Met Pro Asn Val TrpLys Gly Val Val Ala Asp Arg Gly Tyr Leu Glu 195 200 205 Lys Ala Leu AspMet Phe Arg Ala Arg Tyr Ser Ser Pro Val Phe Val 210 215 220 Val Thr SerAsn Gly Met Ala Trp Cys Arg Glu Asn Ile Asn Ala Ser 225 230 235 240 ArgGly Asp Val Val Phe Ala Gly Asn Gly Ile Glu Gly Ser Pro Ala 245 250 255Lys Asp Phe Ala Leu Leu Thr Gln Cys Asn His Thr Ile Met Thr Ile 260 265270 Gly Thr Phe Gly Ile Trp Ala Ala Tyr Leu Ala Gly Gly Asp Thr Ile 275280 285 Tyr Leu Ala Asn Tyr Thr Leu Pro Asp Ser Pro Phe Leu Lys Val Phe290 295 300 Lys Pro Glu Ala Ala Phe Leu Pro Glu Trp Val Gly Ile Pro AlaAsp 305 310 315 320 Leu Ser Pro Leu Leu Lys Ala Leu Thr Pro Ala Cys ProArg Ser His 325 330 335 Phe His Leu Lys Ala Lys Gly Val Thr Cys Tyr ValAla Gly Arg Ala 340 345 350 Phe 11 344 PRT Homo sapiens 11 Met Leu ValVal Gln Met Pro Phe Ser Phe Pro Met Ala His Phe Ile 1 5 10 15 Leu PheVal Phe Thr Val Ser Thr Ile Phe His Val Gln Gln Arg Leu 20 25 30 Ala LysIle Gln Ala Met Trp Glu Leu Pro Val Gln Ile Pro Val Leu 35 40 45 Ala SerThr Ser Lys Ala Leu Gly Pro Ser Gln Leu Arg Gly Met Trp 50 55 60 Thr IleAsn Ala Ile Gly Arg Leu Gly Asn Gln Met Gly Glu Tyr Ala 65 70 75 80 ThrLeu Tyr Ala Leu Ala Lys Met Asn Gly Arg Pro Ala Phe Ile Pro 85 90 95 AlaGln Met His Ser Thr Leu Ala Pro Ile Phe Arg Ile Thr Leu Pro 100 105 110Val Leu His Ser Ala Thr Ala Ser Arg Ile Pro Trp Gln Asn Tyr His 115 120125 Leu Asn Asp Trp Met Glu Glu Glu Tyr Arg His Ile Pro Pro Gly Glu 130135 140 Tyr Val Arg Phe Thr Gly Tyr Pro Cys Ser Trp Thr Phe Tyr His His145 150 155 160 Leu Arg Gln Glu Ile Leu Gln Glu Phe Thr Leu His Asp HisVal Arg 165 170 175 Glu Glu Ala Gln Lys Phe Leu Arg Gly Leu Gln Val AsnGly Ser Arg 180 185 190 Pro Gly Thr Phe Val Gly Val His Val Arg Arg GlyAsp Tyr Val His 195 200 205 Val Met Pro Lys Val Trp Lys Gly Val Val AlaAsp Arg Arg Tyr Leu 210 215 220 Gln Gln Ala Leu Asp Trp Phe Arg Ala ArgTyr Ser Ser Leu Ile Phe 225 230 235 240 Val Val Thr Ser Asn Gly Met AlaTrp Cys Arg Glu Asn Ile Asp Thr 245 250 255 Ser His Gly Asp Val Val PheAla Gly Asp Gly Ile Glu Gly Ser Pro 260 265 270 Ala Lys Asp Phe Ala LeuLeu Thr Gln Cys Asn His Thr Ile Met Thr 275 280 285 Ile Gly Thr Phe GlyIle Trp Ala Ala Tyr Leu Thr Gly Gly Asp Thr 290 295 300 Ile Tyr Leu AlaAsn Tyr Thr Leu Pro Asp Ser Pro Phe Leu Lys Ile 305 310 315 320 Phe LysPro Glu Ala Ala Phe Leu Pro Glu Trp Thr Gly Ile Ala Ala 325 330 335 AspLeu Ser Pro Leu Leu Lys His 340 12 100 DNA Homo sapiens 12 tgtcctctctgtaatcttct tcctccatat ccatcaagac agctttccac atggcctagg 60 cctgtcgatcctgtgtcaag accgccgcct ggtgacaccc 100 13 50 DNA Homo sapiens 13accccaatgg ccggtttggt aatcagatgg gacagtatgc cacgctgctg 50 14 100 DNAHomo sapiens 14 atggacagga ggctacaccg tggaaagact ttgccctgct cacacagtgcaaccacacca 60 ttatgaccat tggcaccttc ggcttctggg ctgcctacct 100 15 100 DNAHomo sapiens 15 catgctggtc gttcagatgc ctttctcctt tcccatggcc cacttcatcctctttgtctt 60 tacggtttcc actatatttc acgttcagca gcggctagcg 100 16 50 DNAHomo sapiens 16 atgcaatagg ccgcctgggg aaccagatgg gcgagtacgc cacactgtac50 17 100 DNA Homo sapiens 17 atggacagga ggctacaccg tggaaagactttgccctgct cacacagtgc aaccacacca 60 ttatgaccat tggcaccttc ggcttctgggctgcctacct 100 18 94 DNA Homo sapiens 18 ccccacagcc gtcaagggattctgggccac ccgcccttcc ttctccacct tctacttcgt 60 ctttgccatt tttgtggtgtccaccatctt tcac 94 19 50 DNA Homo sapiens 19 actccaaggg ccgcctggggaaccagatgg gcgagtacgc cacgctgtac 50 20 100 DNA Homo sapiens 20atggcctcca gggctcacct gccaaggact tcgcactgct cacacagtgc aaccacacca 60tcatcaccgt gggcaccttc ggggtctggg ccgcgtacct 100 21 100 DNA Oryctolaguscuniculus 21 tgccctctct gccttctcct tcctcctgca tctccaccaa gacctctcccgaaacggcct 60 agccctgtct ctcccgtgtc tggaacgcca gccggtgcca 100 22 50 DNAOryctolagus cuniculus 22 acccggatgg ccgctttggg aaccagatgg ggcagtacgccactctgctc 50 23 100 DNA Oryctolagus cuniculus 23 acggcctcga gagctcgccggccaaggact ttgcgctgct cacgcagtgt aaccacaccg 60 tcatgaccat cggcacctttggcttctggg ccgcctacct 100 24 94 DNA Oryctolagus cuniculus 24 tcccacagccaccaggagat tgagggccac ccacccgtcc gtctccacca tctacttcct 60 gttcaccatctttgtggtat ccactgtctt ccac 94 25 50 DNA Oryctolagus cuniculus 25acgccatggg ccgcctgggg aaccagatgg gcgagtacgc cacgctgtac 50 26 100 DNAOryctolagus cuniculus 26 acggcctcga gggctctccg gccaaggact ttgcgctgctcacgcagtgt aaccacaccg 60 tcatgaccat cggcaccttt ggcttctggg ccgcctacct 10027 79 DNA Oryctolagus cuniculus 27 catggtccac gtcatcctct tcgtcttcaccgcctccacc atcttccacc tccagcagcg 60 cctggtgagg attcaaccc 79 28 50 DNAOryctolagus cuniculus 28 acgccatggg ccgcctgggg aaccagatgg gcgagtacgccacgctgtat 50 29 100 DNA Oryctolagus cuniculus 29 atggcctcga gagctcgccggccaaggact ttgcgctgct cacgcaggtt aaccacaccg 60 tcatgaccat cggcacctttgggatctggg ccgcctacct 100

What is claimed is:
 1. An isolated protein comprising an amino acidsequence as depicted in FIG. 5 (SEQ ID NO:8).
 2. An isolated proteincomprising an amino acid sequence as depicted in FIG. 3A (SEQ ID NO:10).3. An isolated protein consisting of an amino acid sequence as depictedin FIG. 5 (SEQ ID NO:8)
 4. An isolated protein consisting of an aminoacid sequence as depicted in FIG. 3A (SEQ ID NO:10).
 5. An isolatedprotein, the amino acid sequence of which consists of a catalytic domaindefined by amino acids numbers 28-380 as depicted in FIG. 5 (SEQ IDNO:8) or amino acids numbers 1-353 as depicted in FIG. 3A (SEQ IDNO:10).
 6. A chimeric protein comprising the protein of claim 3 fused bya covalent bond to at least a portion of a second protein, which secondprotein is not said protein defined by the sequence as depicted in FIG.5 (SEQ ID NO:8).
 7. A chimeric protein according to claim 6 whereinsecond protein is protein A and which portion is the IgG binding domain.8. A chimeric protein comprising the protein of claim 4 or 5 fused by acovalent bond to at least a portion of a second protein, which secondprotein is not said protein defined by the sequence as depicted in FIG.5 (SEQ ID NO:8).
 9. A chimeric protein according to claim 8 whereinsecond protein is protein A and which portion is the IgG binding domain.10. An isolated nucleic acid comprising a nucleotide sequence asdepicted in FIG. 5 (SEQ ID NO:7).
 11. An isolated nucleic acidcomprising a nucleotide sequence as depicted in FIG. 3A (SEQ ID NO:9).12. An isolated nucleic acid comprising a nucleotide sequence encodingan amino acid sequence as depicted in FIG. 5 (SEQ ID NO:8) or itsreverse complement.
 13. An isolated nucleic acid comprising a nucleotidesequence encoding an amino acid sequence as depicted in FIG. 3A (SEQ IDNO:10) or its reverse complement.
 14. An isolated RNA moleculecomprising a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7),wherein the base U (uracil) is substituted for the base T (thymine) ofsaid sequence.
 15. An isolated RNA molecule comprising a nucleotidesequence as depicted in FIG. 3A (SEQ ID NO:9), wherein the base U(uracil) is substituted for the base T (thymine) of said sequence. 16.An isolated RNA molecule comprising a nucleotide sequence encoding anamino acid sequence as depicted in FIG. 5 (SEQ ID NO:8).
 17. An isolatedRNA molecule comprising a nucleotide sequence encoding an amino acidsequence as depicted in FIG. 3A (SEQ ID NO:10).
 18. A vector comprising:(a) a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7); and (b)an origin of replication.
 19. The vector of claim 18 wherein thenucleotide sequence is operably linked to a heterologous promoter.
 20. Avector comprising: (a) a nucleotide sequence as depicted in FIG. 3A (SEQID NO:9); and (b) an origin of replication.
 21. The vector of claim 20wherein the nucleotide sequence is operably linked to a heterologouspromoter.
 22. A vector comprising: (a) a nucleotide sequence that is thereverse complement to all or a fragment of the nucleotide sequence asdepicted in FIG. 5 (SEQ ID NO:7); and (b) an origin of replication. 23.The vector of claim 22 wherein the nucleotide sequence is operablylinked to a heterologous promoter.
 24. A vector comprising: (a) anucleotide sequence encoding an amino acid sequence as depicted in FIG.5 (SEQ ID NO:8) and (b) an origin of replication.
 25. A vectorcomprising: (a) a nucleotide sequence encoding an amino acid sequence asdepicted in FIG. 3A (SEQ ID NO:10) and (b) an origin of replication. 26.A recombinant cell containing a recombinant nucleic acid vectorcomprising a nucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7).27. The recombinant cell of claim 26 wherein the cell is a eukaryoticcell.
 28. The recombinant cell of claim 26 wherein the cell is amammalian cell.
 29. A recombinant cell containing a recombinant nucleicacid vector comprising a nucleotide sequence as depicted in FIG. 3A (SEQID NO:9).
 30. The recombinant cell of claim 29 wherein the cell is aprokaryotic cell.
 31. The recombinant cell of claim 29 wherein the cellis a bacterial cell.
 32. A method of producing a ratα1→2fucosyltransferase protein comprising: (a) culturing a recombinantcell containing the vector of claim 18 such that theα1→2fucosyltransferase protein, encoded by the nucleotide sequence SEQID NO:7 contained in said vector, is expressed by the cell; and (b)recovering the expressed protein or a cellular fraction containing saidprotein.
 33. An isolated or purified protein produced by the method ofclaim
 32. 34. A cellular fraction with protein activity produced by themethod of claim
 32. 35. A method of producing a ratα1→2fucosyltransferase protein comprising: (a) culturing a recombinantcell containing the vector of claim 20 such that theα1→2fucosyltransferase protein, encoded by the nucleotide sequence SEQID NO:9 contained in said vector, is expressed by the cell; and (b)recovering the expressed protein or a cellular fraction containing saidprotein.
 36. An isolated or purified protein produced by the method ofclaim
 35. 37. A cellular fraction with protein activity produced by themethod of claim
 35. 38. A method of producing a ratα1→2fucosyltransferase, protein comprising: (a) culturing a recombinantcell containing the vector of claim 24 such that theα1→2fucosyltransferase protein, encoded by the nucleotide sequence SEQID NO:7 contained in said vector, is expressed by the cell; and (b)recovering the expressed protein or a cellular fraction containing saidprotein.
 39. An isolated or purified protein produced by the method ofclaim
 38. 40. A cellular fraction with protein activity produced by themethod of claim
 38. 41. A method of producing a ratα1→2fucosyltransferase protein comprising: (a) culturing a recombinantcell containing the vector of claim 25 such that theα1→2fucosyltransferase protein, encoded by the nucleotide sequence SEQID NO:9 contained in said vector, is expressed by the cell; and (b)recovering the expressed protein or a cellular fraction containing saidprotein.
 42. An isolated or purified protein produced by the method ofclaim
 41. 43. A cellular fraction with protein activity produced by themethod of claim
 41. 44. A method for detecting the onset of cancercomprising the detection of a nucleotide sequence as depicted in FIG. 5(SEQ ID NO:7) or a fragment or complement thereof.
 45. A method tosuppress or inhibit in a cell the function of an α1→2fucosyltransferaseprotein, said method comprising contacting a cell with a nucleic acidcomprising a nucleotide sequence that is the reverse complement of anucleotide sequence as depicted in FIG. 5 (SEQ ID NO:7) or a fragmentthereof, or as depicted in FIG. 3A (SEQ ID NO:9) or a fragment thereof,and wherein said nucleic acid is RNA, the base T (thymine) in SEQ IDNO:7 and SEQ ID NO:8 is substituted by the base U (uracil).
 46. Themethod of claim 45, wherein said nucleic acid is contained within anadenoviral or retroviral vector.
 47. The method of claim 45, whereinsaid cell is a human small cell lung carcinoma cell.
 48. A method forthe preparative synthesis of a molecule comprising Fucα1→2Galβ1→3GalNAc,said method comprising contacting the isolated or purified ratα1→2fucosyltransferase of claim 1, 2, 3, 4, 5, 6 or 8 with GDP-fucoseand a molecule having a terminal Galβ1→3GalNAc moiety and recovering amolecule comprising Fucα1→2Galβ1→3GalNAc.
 49. A method for thepreparative synthesis of a glycolipid, glycoprotein, glycolipoprotein orfree oligosaccharide comprising Fucα1→2Galβ1→3GalNAc, said methodcomprising contacting the isolated or purified ratα1→2fucosyltransferase of claim 1, 2, 3, 4, 5, 6 or 8 with GDP-fucoseand a glycolipid, glycoprotein, glycolipoprotein or oligosaccharidehaving a terminal Galβ1→3GalNAc moiety and recovering a glycolipid,glycoprotein, glycolipoprotein or free oligosaccharide comprisingFucα1→2Galβ1→3GalNAc.
 50. The method according to claim 49 wherein therat α1→2fucosyltransferase is contacted with an oligosaccharidecomprising a terminal Galβ1→3GalNAc moiety.
 51. A method for thepreparative synthesis of fucosyl-GM₁ comprising contacting the isolatedor purified rat α1→2fucosyltransferase of claim 1, 2, 3, 4, 5, 6 or 8with GDP-fucose and the ganglioside GM₁ and recovering fucosyl-GM₁. 52.A method for the preparative synthesis of a molecule comprisingFucα1→2Galβ1→3GalNAc, said method comprising contacting the isolated orpurified rat α1→2fucosyltransferase of claim 33, 36, 39, or 42 or thecellular fraction of claim 34, 37, 40, or 43 with GDP-fucose and amolecule having a terminal Galβ1→3GalNAc moiety and recovering amolecule comprising Fucα1→2Galβ1→3GalNAc.
 53. A method for thepreparative synthesis of a glycolipid, glycoprotein, glycolipoprotein orfree oligosaccharide comprising Fucα1→2Galβ1→3GalNAc, said methodcomprising contacting the isolated or purified ratα1→2fucosyltransferase of claim 33, 36, 39, or 42 or the cellularfraction of claim 34, 37, 40, or 43 with GDP-fucose and a glycolipid,glycoprotein, glycolipoprotein or oligosaccharide having a terminalGalβ1→3GalNAc moiety and recovering a glycolipid, glycoprotein,glycolipoprotein or free oligosaccharide comprisingFucα1→2Galβ1→3GalNAc.
 54. The method according to claim 53 wherein therat α1→2fucosyltransferase is contacted with an oligosaccharidecomprising a terminal Galβ1→3GalNAc moiety.
 55. A nutritional formulacomposition comprising the glycolipid, glycoprotein, glycolipoprotein oroligosaccharide obtained by the method of claim
 49. 56. A nutritionalformula composition comprising the glycolipid, glycoprotein,glycolipoprotein or oligosaccharide obtained by the method of claim 53.57. A nutritional formula composition comprising the oligosaccharideobtained by the method of claim
 50. 58. A nutritional formulacomposition comprising the oligosaccharide obtained by the method ofclaim
 54. 59. A method for the preparative synthesis of fucosyl-GM₁comprising contacting the isolated or purified ratα1→2fucosyltransferase of claim 33, 36, 39, or 42 or the cellularfraction of claim 34, 37, 40, or 43 with GDP-fucose and the gangliosideGM₁ and recovering fucosyl-GM₁.
 60. A method to induce animmunotherapeutic or immunosuppressive action against afucosyl-GM₁-producing disease, comprising administering fucosyl-GM₁ to ahuman patient with said disease.
 61. The method of claim 60 wherein saiddisease is cancer or neurological disease.
 62. The method of claim 60wherein said disease is small cell lung carcinoma.